DK2986635T3 - EFFECTIVE DELIVERY OF BIG GENES THROUGH DUAL-AAV VECTORS - Google Patents

Description

DESCRIPTION

TECHNICAL FIELD

[0001] The present invention relates to constructs, vectors, relative host cells and pharmaceutical compositions which allow an effective gene therapy, in particular of genes larger than 5Kb.

BACKGROUND OF THE INVENTION

[0002] Inherited retinal degenerations (IRDs), with an overall global prevalence of 1/2,000 (7), are a major cause of blindness worldwide. Among the most frequent and severe IRDs are retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and Stargardt disease (STGD), which are most often inherited as monogenic conditions. The majority of mutations causing IRDs occur in genes expressed in neuronal photoreceptors (PR), rods and/or cones in the retina (2). No therapy is currently available for these blinding diseases.

[0003] Gene therapy holds great promise for the treatment of IRDs. Among the available gene transfer vectors, those based on the small adeno-associated virus (AAV) are most efficient at targeting both PR and retinal pigment epithelium (RPE) (3-4) for long-term treatment upon a single subretinal administration (3-4). Recently the inventors and others, have demonstrated that subretinal administration of AAV is well-tolerated and effective for improving vision in patients affected with type 2 LCA, which is caused by mutations in RPE65, a gene expressed in the RPE (5-9). These results bode well for the treatment of other forms of LCA and IRDs in general. The availability of AAV vector serotypes such as AAV2/8, which efficiently targets PR (10-14) and RPE, further supports this approach. However, a major limitation of AAV is its cargo capacity, which is thought to be limited to around 5 kb, the size of the parental viral genome (75-79). This limits the application of AAV gene therapy approaches for common IRDs that are caused by mutations in genes whose coding sequence (CDS) is larger than 5 kb (herein referred to as large genes). These include:

[0004] Stargardt disease (STGD; MIM#248200) is the most common form of inherited macular degeneration caused by mutations in the ABCA4 gene (CDS: 6822 bp), which encodes the all-trans retinal transporter located in the PR outer segment (20); Usher syndrome type IB (USH1B; MIM#276900) is the most severe form of RP and deafness caused by mutations in the MYO7A gene (CDS: 6648 bp) (27) encoding the unconventional MYO7A, an actin-based motor expressed in both PR and RPE within the retina (22-24).

[0005] Cone-rod dystrophy type 3, fundus flavimaculatus, age-related macular degeneration type 2, Early-onset severe retinal dystrophy, and Retinitis pigmentosa type 19 are also associated with ABCA4 mutations (ABCA4-associated diseases).

[0006] Various strategies have been investigated to overcome the limitation of AAV cargo capacity. Several groups, including the inventors' own, have attempted to "force" large genes into one of the many AAV caspids available by developing the so-called oversize vectors (25-27). Although administration of oversize AAV vectors achieves therapeutically-relevant levels of transgene expression in rodent and canine models of human inherited diseases (27-30), including the retina of the Abca4-/- and shaker 1 (sh1) mouse models of STGD and USH1B (27, 30), the mechanism underlying oversize AAV-mediated transduction remains elusive. In contrast to what the inventors and others originally proposed (25-27), oversize AAV vectors do not contain a pure population of intact large size genomes but rather a heterogeneous mixture of mostly truncated genomes <5kb in length (75-78). Following infection, reassembly of these truncated genomes in the target cell nucleus has been proposed as a mechanism for oversize AAV vector transduction (75-77, 37). Independent of transduction mechanism and in vivo efficacy, the heterogeneity in oversize AAV genome sizes is a major limitation for their application in human gene therapy.

[0007] Alternatively, the inherent ability of AAV genomes to undergo intermolecular concatemerization (32) is exploited to transfer large genes in vivo by splitting a large gene expression cassette into halves (<5kb in size), each contained in one of two separate (dual) AAV vectors (33-35). In the dual AAV trans-splicing strategy, a splice donor (SD) signal is placed at the 3' end of the 5'-half vector and a splice acceptor (SA) signal is placed at the 5'end of the 3'-half vector. Upon co-infection of the same cell by the dual AAV vectors and inverted terminal repeat (ITR)-mediated head-to-tail concatemerization of the two halves, trans-splicing results in the production of a mature mRNA and full-size protein (33). Transsplicing has been successfully used to express large genes in muscle and retina (36-37).

[0008] In particular, Reich et al. (37) used the trans-splicing strategy with AAV2 and AAV5

capsids and show that both vectors transduce both retinal pigment epithelium and photoreceptors using LacZ gene as a reporter gene. This strategy was not employed using a therapeutic and/or large gene.

[0009] Alternatively, the two halves of a large transgene expression cassette contained in dual AAV vectors may contain homologous overlapping sequences (at the 3' end of the 5'-half vector and at the 5' end of the 3'-half vector, dual AAV overlapping), which will mediate reconstitution of a single large genome by homologous recombination (34). This strategy depends on the recombinogenic properties of the transgene overlapping sequences (38). A third dual AAV strategy (hybrid) is based on adding a highly recombinogenic region from an exogenous gene [i.e. alkaline phosphatase, AP (35, 39)] to the trans-splicing vector. The added region is placed downstream of the SD signal in the 5'-half vector and upstream of the SA signal in the 3'-half vector in order to increase recombination between the dual AAVs. The document US2010/003218 is directed to an AP-based hybrid dual vector system. The document shows the transduction efficiency of the AP-based hybrid dual vector expressing mini-dystrophin but no data concerning efficacy.

[0010] Lopes et al. (30) studied retinal gene therapy with a large MYO7A cDNA using adeno-associated virus and found that MYO7A therapy with AAV2 or AAV5 single vectors is efficacious to some extent, while the dual AAV2 approach proved to be less effective. Therefore there is still the need for constructs and vectors that can be exploited to reconstitute large gene expression for an effective gene therapy.

STATEMENT OF FUNDING

[0011] This invention was made with the support of the Italian Telethon Foundation (grant TGM11MT1 and European funds). The Italian Telethon Foundation has rights in this invention.

[0012] Studies on the dual AAV trans-splicing and dual AAV hybrid AP strategies were made with U.S. Government support under Contract No. R24RY019861 awarded by the National Eye Institute. The U.S. Government has certain rights in this invention.

SUMMARY OF THE INVENTION

[0013] Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV cargo capacity limited to 5 kb prevents it from being applied to therapies of those inherited retinal diseases, such as Stargardt disease (STGD) or Usher syndrome type IB (USH1B) that are due to mutations of genes exceeding 5 kb. Previous methods for large gene transfer tested in the retina and based on "forced" packaging of large genes into AAV capsids (oversize AAV) may not be easily translated to the clinical arena due to the heterogeneity of vector genome size, which represents a safety concern.

[0014] Taking advantage of AAV ability to undergo intermolecular concatemerization, the inventors generated dual AAV vectors which reconstitute a large gene by either splicing (transsplicing), homologous recombination (overlapping), or a combination of the two (hybrid).

[0015] To determine which AAV-based strategy most efficiently transduces large genes in the retina, the inventors compared several AAV-based strategies side-by-side in HEK293 cells and in mouse and pig retina in vivo using EGFP, ABCA4 or MYO7A.

[0016] The inventors found that dual trans-splicing and hybrid but not overlapping AAV vectors transduce efficiently mouse and pig photoreceptors, the major cell target for treatment of inherited retinal degenerations. The levels of retinal transduction by dual trans-splicing or hybrid AAV resulted in a significant improvement of the phenotype of Abca4-/- and sh1 mouse models of STGD and USH1B. Dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.

[0017] It is therefore an embodiment of the present invention a dual construct system to express the coding sequence of a gene of interest in an host cell, said coding sequence consisting of a 5'end portion and of a 3'end portion, said dual construct system comprising: 1. a) a first plasmid comprising in a 5'-3' direction: o a AAV 5'-inverted terminal repeat (5'-ITR) sequence; ° a promoter sequence; ° the 5' end portion of said coding sequence, said 5'end portion being operably linked to and under control of said promoter; o a nucleic acid sequence of a splicing donor signal; and o a AAV 3'-inverted terminal repeat (3'-ITR) sequence; and 2. b) a second plasmid comprising in a 5'-3' direction: o a AAV 5'-inverted terminal repeat (5'-ITR) sequence; ° a nucleic acid sequence of a splicing acceptor signal; o the 3'end of said coding sequence; ° a poly-adenylation signal nucleic acid sequence; and o a AAV 3'-inverted terminal repeat (3'-ITR) sequence, wherein said first plasmid further comprises a nucleic acid sequence of a recombinogenic region in 5' position of the AAV 3'ITR of said first plasmid and in 3' position of the nucleic acid sequence of the splicing donor signal and wherein said second plasmid further comprises the nucleic acid sequence of the recombinogenic region in 3' position of the AAV 5'-ITR of said second plasmid and in 5' position of the nucleic acid sequence of the splicing acceptor signal, wherein the recombinogenic region is a F1 phage recombinogenic region that consists of the sequence: GG G ATTTTG CCG ATTTCG GCCTATTG GTTAAAAAATG AG CTG ATTTAACAAAAATTTAACGC GAATTTTAACAAAAT (SEQ ID NO. 3) or a fragment thereof that maintains the recombinogenic property of SEQ ID No. 3 and wherein upon introduction of said first plasmid and said second plasmid into the host cell, said coding sequence reconstitutes by means of the splicing donor and the splicing acceptor signals.

The dual construct system of the present invention is advantageously exploited to reconstitute large gene expression. When the coding sequence reconstitutes, gene expression occurs.

The recombinogenic region may also be a fragment of SEQ ID NO.3, said fragment maintaining the recombinogenic properties of the full length sequence. Preferably the fragment has 70 %, 75 %, 80%, 85 %, 90 %, 95 % or 99 % identity with SEQ ID NO. 3.

[0018] Still preferably, the nucleotide sequence of the ITRs derives from the same or different AAV serotype.

[0019] Preferably, the 3'-ITR of the first plasmid and the 5'-ITR of the second plasmid are from the same AAV serotype.

[0020] Yet preferably, the 5'-ITR and 3'-ITR of the first plasmid and the 5'-ITR and 3'-ITR of the second plasmid are respectively from different AAV serotypes.

[0021] Preferably, the 5'-ITR of the first plasmid and the 3'-ITR of the second plasmid are from different AAV serotypes.

[0022] Yet preferably the coding sequence is split into the 5' end portion and the 3' end portion at a natural exon-exon junction.

[0023] In a preferred embodiment the nucleic acid sequence of the splicing donor signal consists of the sequence:

GTAAGTATGAAGGTTAGAAGACAGGTTTAAGGAGACCAATAGAAAG:TGGGGTTGTCGAGACA GAGAAGACTCTTGCGTTTCT (SEQ ID No. I).

[0024] In a preferred embodiment the nucleic acid sequence of the splicing acceptor signal consists of the sequence: GATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG (SEQ ID No. 2).

[0025] The spicing acceptor signal and the splicing donor signal may also be chosen by the skilled person in the art among sequences known in the art.

[0026] Spliceosomal introns often reside within the sequence of eukaryotic protein-coding genes. Within the intron, a donor site (5' end of the intron), a branch site (near the 3' end of the intron) and an acceptor site (3' end of the intron) are required for splicing. The splice donor site includes an almost invariant sequence GU at the 5' end of the intron, within a larger, less highly conserved region. The splice acceptor site at the 3' end of the intron terminates the intron with an almost invariant AG sequence. Upstream (5'-ward) from the AG there is a region high in pyrimidines (C and U), or polypyrimidine tract. Upstream from the polypyrimidine tract is the branchpoint, which includes an adenine nucleotide.

[0027] In a preferred embodiment the first plasmid further comprises at least one enhancer sequence, operably linked to the coding sequence. Any known suitable enhancer sequence may be selected by the skilled person in the art.

[0028] Preferably the coding sequence is a nucleotide sequence encoding a protein able to correct a genetic disease, in particular an inherited retinal degeneration.

[0029] Still preferably the coding sequence is selected from the group consisting of: ABCA4, MYO7A, CEP290, CDH23, EYS, USH2a, GPR98 or ALMS1.

[0030] It is a further embodiment of the invention a dual adeno-associated virus (AAV) viral vector system comprising: 1. a) a first AAV viral vector containing the first plasmid comprising in a 5'-3' direction: a 5'-inverted terminal repeat (5'-ITR) sequence, a promoter sequence, the 5' end portion of said coding sequence, said 5'end portion being operably linked to and under control of said promoter, a nucleic acid sequence of a splicing donor signal, and a 3'-inverted terminal repeat (3'-ITR) sequence; and 2. b) a second AAV viral vector containing the second plasmid comprising in a 5'-3' direction: a 5'-inverted terminal repeat (5'-ITR) sequence, a nucleic acid sequence of a splicing acceptor signal, the 3'end of said coding sequence, a poly-adenylation signal nucleic acid sequence; and a 3'-inverted terminal repeat (3'-ITR) sequence.

[0031] Still preferably the adeno-associated virus (AAV) vectors are selected from the same or different AAV serotypes.

[0032] Still preferably the adeno-associated virus is selected from the serotype 2, the serotype 8, the serotype 5, the serotype 7 or the serotype 9.

[0033] It is a further embodiment of the invention a host cell comprising the dual viral vector system according to the invention.

[0034] Preferably the host cell is a mammalian cell, a human cell, a retinal cell, a non-embryonic stem cell.

[0035] It is a further embodiment of the invention the dual construct system of the invention, the dual viral vector system of the invention or the host cell of the invention for medical use, preferably for use in a gene therapy, still preferably for the treatment and/or prevention of a pathology or disease characterized by a retinal degeneration. Preferably, the retinal degeneration is inherited.

[0036] Still preferably the pathology or disease is selected from the group consisting of: retinitis pigmentosa, Leber congenital amaurosis (LCA), Stargardt disease, Usher disease, Alstrom syndrome, a disease caused by a mutation in the ABCA4 gene (also named a ABCA4-associated disease). Cone-rod dystrophy type 3, fundus flavimaculatus, age-related macular degeneration type 2, Early-onset severe retinal dystrophy, and Retinitis pigmentosa type 19 are examples of disease caused by a mutation in the ABCA4 gene (ABCA4-associated diseases).

[0037] It is a further embodiment of the invention a pharmaceutical composition comprising the dual construct system according to the invention, the dual viral vector system according to the invention or the host cell according to the invention and pharmaceutically acceptable vehicle. It is a further embodiment of the invention a method for treating and/or preventing a pathology or disease characterized by a retinal degeneration comprising administering to a subject in need thereof an effective amount of the dual construct system as described herein , the dual viral vector system as described herein or the host cell as described herein .

[0038] It is a further embodiment of the invention a nucleic acid consisting of SEQ ID No. 3 for use as a recombinogenic region.

[0039] It is a further embodiment of the invention an in vitro method to induce genetic recombination comprising using the sequence consisting of SEQ ID No. 3 and the sequence consisting of SEQ ID No. 3 for use in a method to induce genetic recombination.

[0040] In the present invention preferably the promoter is selected from the group consisting of: cytomegalovirus promoter, Rhodopsin promoter, Rhodopsin kinase promoter, Interphotoreceptor retinoid binding protein promoter, vitelliform macular dystrophy 2 promoter. However any suitable promoter known in the art may be used.

[0041] In the present invention, the coding sequence is split into a first and a second fragment (5' end portion and 3' end portion) at a natural exon-exon junction. Preferably each fragment of the coding sequence should not exceed a size of 10 kb. Preferably each 5' end portion and 3' end portion may have a size of 4.5Kb, 5Kb, 5.5 Kb, 6Kb, 6.5 Kb, 7kb, 7.5 Kb, 8 Kb, 8.5 Kb, 9Kb, 9.5 Kb or a smaller size.

[0042] During the past decade, gene therapy has been applied to the treatment of disease in hundreds of clinical trials. Various tools have been developed to deliver genes into human cells; among them, genetically engineered viruses, including adenoviruses, are currently amongst the most popular tool for gene delivery. Most of the systems contain vectors that are capable of accommodating genes of interest and helper cells that can provide the viral structural proteins and enzymes to allow for the generation of vector-containing infectious viral particles. Adeno-associated virus is a family of viruses that differs in nucleotide and amino acid sequence, genome structure, pathogenicity, and host range. This diversity provides opportunities to use viruses with different biological characteristics to develop different therapeutic applications. As with any delivery tool, the efficiency, the ability to target certain tissue or cell type, the expression of the gene of interest, and the safety of adenoviral-based systems are important for successful application of gene therapy. Significant efforts have been dedicated to these areas of research in recent years. Various modifications have been made to Adeno-associated virus-based vectors and helper cells to alter gene expression, target delivery, improve viral titers, and increase safety. The present invention represents an improvement in this design process in that it acts to efficiently deliver genes of interest into such viral vectors. Viruses are logical tools for gene delivery. They replicate inside cells and therefore have evolved mechanisms to enter the cells and use the cellular machinery to express their genes. The concept of virus-based gene delivery is to engineer the virus so that it can express the gene of interest. Depending on the specific application and the type of virus, most viral vectors contain mutations that hamper their ability to replicate freely as wild-type viruses in the host. Viruses from several different families have been modified to generate viral vectors for gene delivery. These viruses include retroviruses, lentivirus, adenoviruses, adeno-associated viruses, herpes simplex viruses, picornaviruses, and alphaviruses. The present invention preferably employs adeno-associated viruses.

[0043] An ideal adeno-associated virus based vector for gene delivery must be efficient, cell-specific, regulated, and safe. The efficiency of delivery is important because it can determine the efficacy of the therapy. Current efforts are aimed at achieving cell-type-specific infection and gene expression with adeno-associated viral vectors. In addition, adeno-associated viral vectors are being developed to regulate the expression of the gene of interest, since the therapy may require long-lasting or regulated expression. Safety is a major issue for viral gene delivery because most viruses are either pathogens or have a pathogenic potential. It is important that during gene delivery, the patient does not also inadvertently receive a pathogenic virus that has full replication potential.

[0044] Adeno-associated virus (AAV) is a small virus which infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy, and for the creation of isogenic human disease models.

[0045] Wild-type AAV has attracted considerable interest from gene therapy researchers due to a number of features. Chief amongst these is the virus's apparent lack of pathogenicity. It can also infect non-dividing cells and has the ability to stably integrate into the host cell genome at a specific site (designated AAVS1) in the human chromosome 19. The feature makes it somewhat more predictable than retroviruses, which present the threat of a random insertion and of mutagenesis, which is sometimes followed by development of a cancer. The AAV genome integrates most frequently into the site mentioned, while random incorporations into the genome take place with a negligible frequency. Development of AAVs as gene therapy vectors, however, has eliminated this integrative capacity by removal of the rep and cap from the DNAof the vector. The desired gene together with a promoter to drive transcription of the gene is inserted between the inverted terminal repeats (ITR) that aid in concatamer formation in the nucleus after the single-stranded vector DNA is converted by host cell DNA polymerase complexes into double-stranded DNA. AAV-based gene therapy vectors form episomal concatamers in the host cell nucleus. In non-dividing cells, these concatemers remain intact for the life of the host cell. In dividing cells, AAV DNA is lost through cell division, since the episomal DNA is not replicated along with the host cell DNA. Random integration of AAV DNA into the host genome is detectable but occurs at very low frequency. AAVs also present very low immunogenicity, seemingly restricted to generation of neutralizing antibodies, while they induce no clearly defined cytotoxic response. This feature, along with the ability to infect quiescent cells present their dominance over adenoviruses as vectors for the human gene therapy. AAV genome, transcriptome and proteome [0046] The AAV genome is built of single-stranded deoxyribonucleic acid (ssDNA), either positive-or negative-sensed, which is about 4.7 kilobase long. The genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The former is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry. ITR sequences [0047] The Inverted Terminal Repeat (ITR) sequences comprise 145 bases each. They were named so because of their symmetry, which was shown to be required for efficient multiplication of the AAV genome. Another property of these sequences is their ability to form a hairpin, which contributes to so-called self-priming that allows primase-independent synthesis of the second DNA strand. The ITRs were also shown to be required for both integration of the AAV DNA into the host cell genome (19th chromosome in humans) and rescue from it, as well as for efficient encapsidation of the AAV DNA combined with generation of a fully assembled, deoxyribonuclease-resistant AAV particles.

[0048] With regard to gene therapy, ITRs seem to be the only sequences required in cis next to the therapeutic gene: structural (cap) and packaging (rep) genes can be delivered in trans. With this assumption many methods were established for efficient production of recombinant AAV (rAAV) vectors containing a reporter or therapeutic gene. However, it was also published that the ITRs are not the only elements required in cis for the effective replication and encapsidation. A few research groups have identified a sequence designated cis-acting Rep-dependent element (CARE) inside the coding sequence of the rep gene. CARE was shown to augment the replication and encapsidation when present in cis.

[0049] As of 2006 there have been 11 AAV serotypes described, the 11th in 2004. All of the known serotypes can infect cells from multiple diverse tissue types. Tissue specificity is determined by the capsid serotype and pseudotyping of AAV vectors to alter their tropism range will likely be important to their use in therapy. In the present invention ITRs of AW serotype 2 and serotype 5 are prefered.

Serotype 2 [0050] Serotype 2 (AAV2) has been the most extensively examined so far. AAV2 presents natural tropism towards skeletal muscles, neurons, vascular smooth muscle cells and hepatocytes.

[0051] Three cell receptors have been described for AAV2: heparan sulfate proteoglycan (HSPG), avP5 integrin and fibroblast growth factor receptor 1 (FGFR-1). The first functions as a primary receptor, while the latter two have a co-receptor activity and enable AAV to enter the cell by receptor-mediated endocytosis. These study results have been disputed by Qiu, Handa, et al.. HSPG functions as the primary receptor, though its abundance in the extracellular matrix can scavenge AAV particles and impair the infection efficiency.

Serotype 2 and cancer [0052] Studies have shown that serotype 2 of the virus (AAV-2) apparently kills cancer cells without harming healthy ones. "Our results suggest that adeno-associated virus type 2, which infects the majority of the population but has no known ill effects, kills multiple types of cancer cells yet has no effect on healthy cells," said Craig Meyers, a professor of immunology and microbiology at the Penn State College of Medicine in Pennsylvania. This could lead to a new anti-cancer agent.

Other Serotypes [0053] Although AAV2 is the most popular serotype in various AAV-based research, it has been shown that other serotypes can be more effective as gene delivery vectors. For instance AAV6 appears much better in infecting airway epithelial cells, AAV7 presents very high transduction rate of murine skeletal muscle cells (similarly to AAV1 and AAV5), AAV8 is superb in transducing hepatocytes and AAV1 and 5 were shown to be very efficient in gene delivery to vascular endothelial cells. In the brain, most AAV serotypes show neuronal tropism, while AAV5 also transduces astrocytes. AAV6, a hybrid of AAV1 and AAV2, also shows lower immunogenicity than AAV2.

[0054] Serotypes can differ with the respect to the receptors they are bound to. For example AAV4 and AAV5 transduction can be inhibited by soluble sialic acids (of different form for each of these serotypes), and AAV5 was shown to enter cells via the platelet-derived growth factor receptor.

[0055] In the present invention the delivery vehicles of the present invention may be administered to a patient. A skilled worker would be able to determined appropriate dosage rates. The term "administered" includes delivery by viral or non-viral techniques. Viral delivery mechanisms include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors etc as described above. Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.

[0056] The delivery of one or more therapeutic genes by a vector system according to the present invention may be used alone or in combination with other treatments or components of the treatment.

[0057] The present invention also provides a pharmaceutical composition for treating an individual by gene therapy, wherein the composition comprises a therapeutically effective amount of the vector/construct or host cell of the present invention comprising one or more deliverable therapeutic and/or diagnostic transgenes(s) or a viral particle produced by or obtained from same. The pharmaceutical composition may be for human or animal usage. Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular individual. The composition may optionally comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), and other carrier agents that may aid or increase the viral entry into the target site (such as for example a lipid delivery system). Where appropriate, the pharmaceutical compositions can be administered by any one or more of: inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

[0058] The man skilled in the art is well aware of the standard methods for incorporation of a polynucleotide or vector into a host cell, for example transfection, lipofection, electroporation, microinjection, viral infection, thermal shock, transformation after chemical permeabilisation of the membrane or cell fusion.

[0059] As used herein, the term "host cell or host cell genetically engineered" relates to host cells which have been transduced, transformed or transfected with the construct or with the vector described previously.

[0060] As representative examples of appropriate host cells, one can cites bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, insect cells such as Sf9, animal cells such as CHO or COS, plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. Preferably, said host cell is an animal cell, and most preferably a human cell. The invention further provides a host cell comprising any of the recombinant expression vectors described herein. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for instance, DH5a, E. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like.

[0061] The present invention will now be illustrated by means of non-limiting examples in reference to the following drawings.

Figure 1. Schematic representation of AAV-based strategies for large gene transduction. CDS: coding sequence; pA: poly-adenilation signal; SD: splicing donor signal; SA: splicing acceptor signal; AP: alkaline phosphatase recombinogenic region (39); AK: F1 phage recombinogenic region. Dotted lines show the splicing occurring between SD and SA, pointed lines show overlapping regions available for homologous recombination. The inventors found that dual trans-splicing and hybrid AK may be used to successfully reconstitute large gene expression. In particular dual trans-splicing and hybrid AK vectors, but not overlapping and hybrid AP vectors, transduce efficiently mouse and pig photoreceptors. Normal size and oversize AAV vector plasmids contained full length expression cassettes including the promoter, the full-length transgene CDS and the poly-adenilation signal (pA) (Table 1). The two separate AAV vector plasmids (5' and 3') required to generate dual AAV vectors contained either the promoter followed by the N-terminal portion of the transgene CDS (5' plasmid) or the C-terminal portion of the transgene CDS followed by the pA signal (3' plasmid, Table 1). The structure of all plasmids is indicated in the material and method section.

Figure 2. Dual AAV overlapping, trans-splicing and hybrid AK vectors efficiently transduce large genes in vitro.

Western blot of HEK293 cells infected with AAV2/2 vectors encoding for EGFP (A and D), ABCA4 (B and E) and MYO7A (C and F). (A to C) The arrows indicate full-length proteins, the micrograms of proteins loaded are depicted under each lane, the molecular weight ladder is depicted on the left. (D to F) Quantification of EGFP (D), ABCA4 (E) and MYO7A (F) protein bands. The intensity of the EGFP, ABCA4 and MYO7A bands was divided by the intensity of the Tubulin (D) or Filamin A (E-F) bands. The histograms show the expression of proteins as a percentage relative to dual AAV trans-splicing (TS) vectors, the mean value is depicted above the corresponding bar. Error bars: mean ± s.e.m. (standard error of the mean). (A-C) The Western blot images are representative of and the quantifications are from n=4 (A-B) or n=3 (C) independent experiments. OZ: AAV oversize; OV: dual AAV overlapping; TS: dual AAV trans-splicing; AP: dual AAV hybrid AP; AK: dual AAV hybrid AK; 5'+3': cells co-infected with 5'-and 3'- half vectors; 5': control cells infected with the 5'- half vector only; 3': control cells infected with the 3'-half only; α-EGFP: anti-EGFP antibody; a-3xflag: anti-3xflag antibody; a-MY07A: anti-MYO7A antibody; α-β-Tubulin: anti-P-tubulin antibody; α-Filamin A: anti-filamin A antibody. * ANOVA p value<0.05; ** ANOVA p value< 0.001. (F) The asterisks depicted in the lower panel represent significant differences with both OZ and AP. In

Figure 3. Dual AAV overlapping vectors transduce RPE but not photoreceptors in the mouse and pig retina.

Western blot analysis of C57BL/6 (A) and Large White pig (B) retinal lysates one month following injection of AAV2/8 dual AAV overlapping vectors encoding for ABCA4-3xflag (OV) or AAV2/8 vectors encoding for normal size EGFP (EGFP), under the control of the ubiquitous cytomegalovirus (CMV) promoter, the PR-specific Rhodopsin (RHO) and Rhodopsin kinase (RHOK) promoters, or the RPE-specific vitelliform macular dystrophy 2 (VMD2) promoter. (A-B) The arrows indicate full-length proteins, the molecular weight ladder is depicted on the left, 150 micrograms of proteins were loaded in each lane. The number (n) and percentage of ABCA4-positive retinas out of total retinas analyzed is depicted; a-3xflag: anti-3xflag antibody; a-Dysferlin: anti-Dysferlin antibody (C) Western blot analysis on C57/BL6 eyecups (left panel) and retinas (right panel) at 3 months following the injection of AAV2/8 overlapping vectors encoding for MYO7A-HA (OV) under the control of the ubiquitous chicken-beta-actin (CBA) promoter or the photoreceptor-specific rhodopsin (RHO) promoter. The arrow points at full-length proteins, the molecular weight ladder is depicted on the left, 100 micrograms of protein were loaded in each lane. The number (n) and percentage of MYO7A positive retinas out of total retinas analyzed is depicted. α-HA: anti-hemagglutinin (HA) antibody.

Figure 4. Dual AAV trans-splicing and hybrid AK vectors efficiently transduce both RPE and photoreceptors.

Fluorescence analysis of retinal cryosections from C57BL/6 mice one month following subretinal injection of AAV2/8 vectors encoding for EGFP under the control of the ubiquitous cytomegalovirus (CMV) promoter. The scale bar (20 pm) is depicted in the figure. NS: AAV normal size; OZ: AAV oversize;; TS: dual AAV trans-splicing; AP: dual AAV hybrid AP; AK: dual AAV hybrid AK; RPE: retinal pigmented epithelium; ONL: outer nuclear layer.

Figure 5. Dual AAV trans-splicing and hybrid AK efficiently transduce mouse and pig photoreceptors. (A) Fluorescence analysis of retinal cryosections from C57BL/6 mice one month following subretinal injection of AAV2/8 vectors encoding for EGFP under the control of the PR-specific Rhodopsin promoter (RHO). The scale bar (20 pm) is depicted in the figure. (B) Fluorescence analysis of retinal cryosections from Large White pigs one month following subretinal injection of AAV2/8 vectors encoding for EGFP under the control of the PR-specific RHO promoter. The scale bar (50 pm) is depicted in the figure. NS: AAV normal size; TS: dual AAV trans-splicing; AK: dual AAV hybrid AK; RPE: retinal pigmented epithelium; ONL: outer nuclear layer.

Figure 6. Subretinal administration of dual AAV trans-splicing and hybrid AK vectors results in robust yet variable levels ofABCA4 expression in mouse photoreceptors. (A) Western blot analysis of C57BL/6 retinal lysates one month following the injection of dual AAV trans-splicing (TS) and dual AAV hybrid AK (AK) vectors encoding for ABCA4 under the control of the PR-specific Rhodopsin promoter (RHO). The arrow points at full-length proteins, the molecular weight ladder is depicted on the left, 150 micrograms of protein were loaded in each lane. The number (n) and percentage of ABCA4-positive retinas out of total retinas analysed is depicted. 5'+3': retinas co-injected with 5'-and 3'-half vectors; a-3xflag: anti-3xflag antibody; α-Dysferlin: anti-Dysferlin antibody. (B) Immuno-electron microscopy analysis with anti-HA antibody of retinal sections from wild-type Balb/C (WT; n=3 eyes) and Abca4-i- mice injected with dual AAV hybrid AK vectors (AK-A6CA4; n=5 eyes) or with AAV normal size EGFP (EGFP, n=3 eyes) as control. The black dots represent the immuno-gold labelling of the ABCA4-HA protein. The scale bar (200 nm) is depicted in the figure.

Figure 7. Subretinal injection of dual AAV hybrid AK vectors reduces accumulation of lipofuscin granules in Abca4-i- mice. (A) Transmission electron microscopy analysis of retinal sections from wild-type Balb/c (WT) and Abca4-i- mice injected with either dual AAV hybrid AK vectors (Abca4-I- AK-ABCA4) or with AAV normal size EGFP (Abca4-I- EGFP) as control. The black arrows indicate lipofuscin granules. The scale bar (1.6 pm) is depicted in the figure. (B) Quantification of the mean number of lipofuscin granules counted in at least 30 fields (25pm2) for each sample. WT: Balb/c mice; Abca4-i- EGFP/5'/3': Abca4-i- mice injected with either AAV normal size EGFP or the 5' or 3' half vector of the dual AAV hybrid AK, as control; Abca4-i- AK-ABCA4'. mice injected with dual AAV hybrid AK vectors; Abca4-i- TS-ABCA4: mice injected with dual AAV transsplicing vectors. The number (n) of eyes analysed is depicted. The mean value is depicted above the corresponding bar. Error bars: mean ± s.e.m. (standard error of the mean). * p ANOVA<0.05

Figure 8. Subretinal injections of dual AAV hybrid AK vectors reduces the thickness of Abca4-ΙΕΡΕ. (A) Representative pictures of transmission electron microscopy analysis of retinal sections from wild-type Balb/c (WT) and Abca4-I- mice injected with either dual AAV trans-splicing (TS-ABCA4) and hybrid AK vectors (AK-ABCA4) or with AAV normal size EGFP (EGFP) and 5' or 3' half of the dual hybrid AK vectors (573') as control. The dotted lines indicate the edges of RPE cells. The scale bar (3.8 pm) is depicted in the figure. (B) Quantification of the mean RPE thickness counted in at least 30 fields for each sample. The number (n) of eyes analysed is depicted. The mean value is depicted above the corresponding bar. Error bars: mean ± s.e.m (standard error of the mean), s.d.m: WT: ±716; TS-A6CA4: ± 698.

Figure 9. Subretinal administration of dual AAV trans-splicing and hybrid AK vectors results in robust MY07A expression in mice.

Western blot analysis of C57BL/6 eyecups one month following the injection of dual AAV transsplicing (TS) and hybrid AK (AK) vectors encoding for MYO7A-HA under the control of the ubiquitous chicken beta-actin (CBA) promoter. The arrow indicates full-length proteins, the molecular weight ladder is depicted on the left, 100 micrograms of proteins were loaded in each lane. The number (n) and percentage of MYO7A-positive eyecups out of total retinas analyzed is depicted. 5'+3': eyes co-injected with 5'- and 3'- half vectors; 5': eyes injected with 5'- half vectors; 3': eyes injected with 3'-half vectors; α-HA: anti-hemagglutinin (HA) antibody; α-Dysferlin: anti-Dysferlin antibody.

Figure 10. Subretinal administration of dual AAV trans-splicing and hybrid AK vectors rescues melanosome localization in sh1-i- RPE. (A) Representative semi-thin retinal sections stained with Epoxy tissue stain of sh1+/+ and sh 1 +/- eyes injected with AAV normal size EGFP (EGFP, n=4 eyes), and of sh1-i- eyes injected with dual AAV trans-splicing (TS-MYO7A, n=3 eyes), hybrid AK (AK-/WYO7A; n=3 eyes) or 5'-half vectors (5'TS/5'AK, n=4 eyes), as control. The scale bar (10 pm) is depicted in the figure. (B) Quantification of melanosome localization in the RPE villi of sh1 mice two months following subretinal delivery of dual AAV vectors. The quantification is depicted as the mean number of apical melanosomes/field, the mean value is depicted above the corresponding bar. Error bars: mean ± s.e.m. (standard error of the mean). * p ANOVA<0.05, ** p ANOVA<0.001.

Figure 11. Subretinal administration of dual AAV trans-splicing and hybrid AK vectors reduces rhodopsin accumulation at sh1-i- PR connecting cilia.

Quantification of the number of rhodopsin gold particles at the PR connecting cilium of sh1 mice two months following subretinal delivery of dual AAV vectors. The quantification is depicted as the mean number of gold particles per length of connecting cilia (nm), the mean value is depicted above the corresponding bar. Error bars: mean ± s.e.m. (standard error of the mean).

Figure 12. Dual AAV trans-splicing and hybrid AK vectors efficiently transduce the large gene CEP290 in vitro.

Western blot of HEK293 cells infected with AAV2/2 vectors encoding for CEP290 tagged at its C-terminus with the hemagglutinin (HA) tag (A-B). (A) The arrow indicate the full-length protein, 60 micrograms of proteins were loaded for each lane, the molecular weight ladder is depicted on the left. (B) Quantification of CEP290 protein bands. The intensity of the CEP290 bands was divided by the intensity of the Filamin A bands. The histogram shows the expression of proteins as a percentage relative to dual AAV trans-splicing (TS) vectors, the mean value is depicted above the corresponding bar. Error bars: mean ± s.e.m. (standard error of the mean). The Western blot image is representative of and the quantification is from n=5 independent experiments. OV: dual AAV overlapping; TS: dual AAV trans-splicing; AK: dual AAV hybrid AK; 5'+3': cells co-infected with 5'- and 3'- half vectors; 3': control cells infected with the 3'-half only; α-HA: anti-HA antibody; α-Filamin A: anti-filamin A antibody.

Figure 13. Improved recovery from light desensitization in 3 months old Abca4-i- mice treated with dual AAV trans-splicing and hybrid AK vectors

Recovery from light desensitization in Abca4-i- and Balb/c mice at 6 weeks post-injection. The relative b-wave is the ratio between the post- and the pre-desensitization b-wave amplitudes (pV) both evoked by 1 cd s/m2. The time (minutes) refers to the time post-desensitization. The mean recovery (%) at 60 minutes is depicted, p ANOVA Abca4-i- AK-ABCA4 vs Abca4-i- uninjected/5': 0.05; p ANOVA Abca4-/- TS-ABCA4 vs Abca4-/- uninjected/5': 0.009; p ANOVA Abca4-/- AK-ABCA4 vs WT: 0.002; p ANOVA Abca4-/- TS-ABCA4 vs WT: 0.02; p ANOVA WT vs Abca4-/- uninjected/5': 0.00001. WT: Balb/c mice (n=4); Abca4-i- TS-ABCA4: mice injected with dual AAV trans-splicing vectors (n=5); Abca4-i- AK-ABCA4: mice injected with dual AAV hybrid AK vectors (n=5); Abca4-i- uninjected/5’: Abca4-/- mice either not injected (n=2) or injected with the 5' half of the dual AAV TS or hybrid AK vectors (n=5). Data are depicted as mean ± s.e.m (standard error of the mean). * p ANOVA<0.05.

Figure 14. Dual AAV hybrid AK vectors induce stronger MY07A expression than dual AAV trans-splicing vectors in sh1-/- photoreceptors.

Quantification of MYO7A levels from dual AAV vectors in sh1-/- eyes relative to endogenous Myo7a expressed in sh1+/+ eyes. Sh1-/- eyes were injected with dual AAV TS and hybrid AK vectors encoding MYO7A under the control of either the CBA (left panel) or RHO (right panel) promoters. The histograms show the expression of MYO7A protein as percentage relative to sh1+/+ Myo7a; the mean value is depicted above the corresponding bar. The quantification was performed by Western blot analysis using the anti-MYO7A antibody and measurements of MYO7A and Myo7a band intensities normalized to Dysferlin (data not shown). Error bars: mean ± s.d.m. (standard deviation of the mean). The quantification is representative of: i. left panel: n=2 sh1+/+ eyecups, and n=5 or n=1 sh1-/- eyecups treated with either TS-MYO7A or AK-MY07A, respectively; ii. right panel: n=2 sh1+/+ retinas, and n=1 or n=3 sh1-/- retinas treated with either TS-MYO7A or AK-MYO7A, respectively. ** p Student's t-test <0.001.

Figure 15. AAV normal size, dual AAV trans-splicing and hybrid AK vectors provide the most robust transduction following subretinal delivery in mice.

Live-imaging fundus fluorescence of C57BL/6 eyes one month following subretinal injection of AAV2/8 vectors encoding for EGFR NZ: Normal Size; OZ: AAV oversize; TS: dual AAV transsplicing; AP: dual AAV hybrid AP; AK: dual AAV hybrid AK. Each panel shows a different eye.

Figure 16. Robust ABCA4 and MY07A expression following delivery of dual AAV trans-splicing and hybrid AK vectors to the pig retina, (a) Western blot analysis of large white pig retinal lysates 1 month following injection of dual AAV2/8 trans-splicing (TS; n=2) and hybrid AK (AK; n=3) vectors encoding for ABCA4-3xflag or AAV2/8 vectors encoding for NS EGFP (neg), as negative control, under the control of the photoreceptor-specific rhodopsin (RHO) promoter, (b) Western blot analysis of large white pig retinal lysates one month following injection of dual AAV2/8 trans-splicing (TS: n=5 RPE; n=3 retina) and hybrid AK (AK: n=5 RPE, n=5 retina) vectors encoding for MYO7A-HA under the control of the ubiquitous chicken beta actin (CBA) promoter or single 3'-half of dual AAV-MYO7AHA (neg), as negative control, (a-b) The arrows indicate full-length proteins, the molecular weight ladder is depicted on the left, 150-180 pg of proteins were loaded in each lane. a-3xflag, anti-3xflag antibody; α-HA, anti-hemagglutinin antibody; α-dysferlin, anti-dysferlin antibody.

Figure 17. Dual AAV hybrid AK vectors with heterologous ITRs transduce large genes in vitro. (a) Design of dual AAV hybrid AK vectors with heterologous ITR2 and ITR5. (b) Western blot analysis of HEK293 cells infected with dual AAV hybrid AK vectors with heterologous ITRs encoding for ABCA4 (left panel) and MY07A (right panel). The arrows indicate full-length proteins, 50 micrograms of proteins were loaded, the molecular weight ladder is depicted on the left. 5'+3': cells co-infected with 5'- and 3'- half vectors; 5': control cells infected with the 5'-half vector only; 3': control cells infected with the 3'-half vector only; neg: cells infected with AAV2/8 vectors encoding for EGFP. a-3xflag: anti-3xflag antibody; a-MYO7A: anti-MYO7A antibody; α-Filamin: anti-filamin A antibody, (a) Prom: promoter; CDS: coding sequence; pA: poly-adenylation signal; SD: splicing donor signal; SA: splicing acceptor signal; Pointed lines show overlapping regions available for homologous recombination, dotted lines show the splicing occurring between SD and SA. The position of the heterologous ITR2 and ITR5 is depicted.

DETAILED DESCRIPTION OF THE INVENTION

MATERIALS AND METHODS

Generation of AAV vector plasmids [0062] The plasmids used for AAV vector production were derived from either the pZac2.1 (52) or pAAV2.1 (53) plasmids that contain the inverted terminal repeats (ITRs) of AAV serotype 2 (Table 1).

Table 1. Plasmids for AAV vector production.

N.B. CMV: cytomegalovirus promoter; CBA: chicken beta-actin; RHO: human Rhodopsin promoter; RHOK: human Rhodopsin kinase promoter; Vmd2: vitelliform macular dystrophy 2 promoter; EGFP: enhanced green fluorescent protein; ABCA4: human ATP-binding cassette, sub-family A, member 4; MY07A: human MYOSIN VIIA; SV40: simian virus 40 poly-adenilation signal; BGH: bovine growth hormone poly-adenilation signal; 3xflag: 3xflag tag; HA: hemagglutinin tag; AP: alkaline phosphatase recombinogenic region; AK: F1 phage recombinogenic region; TS: trans-splicing; ITR5:2: plasmid with the left ITR from AAV serotype 5 and the right ITR from AAV serotype 2; ITR2:5: plasmid with the left ITR from AAV serotype 2 and the right ITR from AAV serotype 5. When not specified the left and right ITR are from AAV serotype 2.

[0063] Normal size and oversize AAV vector plasmids contained full length expression cassettes including the promoter, the full-length transgene CDS and the poly-adenilation signal (pA) (Table 1). The two separate AAV vector plasmids (5' and 3') required to generate dual AAV vectors contained either the promoter followed by the N-terminal portion of the transgene CDS (5' plasmid) or the C-terminal portion of the transgene CDS followed by the pA signal (3' plasmid, Table 1). Normal size EGFP plasmids were generated by cloning the EGFP CDS of pAAV2.1-CMV-EGFP plasmid (720 bp) (53) in pZac2.1 (52); oversize EGFP was generated from pAAV2.1-CMV-EGFP (53) by inserting a DNA stuffer sequence of 3632 bp from human ABCA4 (NM_000350.2, bp 1960-5591) upstream of the CMV promoter and a second DNA stuffer sequence of 3621 bp, composed of: murine ABCA4 (NM 007378.1, 1066-1 and 7124-6046 bp; 2145 total bp) and human Harmonin (NM153676.3 131-1606 bp; 1476 total bp), downstream of the pA signal (This construct was used in the experiments of Fig. 1a, d, Fig. 4 and Fig. 15). To generate dual AAV vector plasmids, the EGFP CDS (720 bp) was split into two constructs: one containing the N-terminal CDS (PMID: 9759496, bp 1-393) and the other containing the C-terminal CDS (PMID: 9759496, bp 394-720).

[0064] The oversize ABCA4 plasmids contained the full-length human ABCA4 CDS

(GeneNM_000350.2, bp 105-6926), while the oversize MY07A plasmids contained the full-length human MY07A CDS from isoform 1 (NM_000260.3, bp 273-6920). To generate plasmids for dual AAV OV vectors the ABCA4 and MY07A CDS were split into two constructs, one containing N-terminal CDS (ABCA4: NM_000350.2, bp 105-3588; MI07A. NM_000350.2, bp 273-3782) and the other containing C-terminal CDS (ABCA4'. NM_000350.2, bp 2819-6926; MYO7A: NM_000350.2, bp 2913-6920). Therefore, the region of homology shared by overlapping vector plasmids was 770bp for ABCA4 and 870 bp for MYO7A. To generate plasmids for dual AAV OV vectors the human CEP290 CDS was split into two constructs, one containing N-terminal CDS (CEP290: NM_025114,bp 345-4076) and the other containing C-terminal CDS (CEP290: NM_025114, bp 3575-7784). Therefore, the region of homology shared by overlapping vector plasmids was 502 bp.

[0065] To generate trans-splicing and hybrid vector plasmids the ABCA4 and MY07A CDS were split at a natural exon-exon junction. ABCA4 was split between exons 19-20 (5' half: NM_000350.2, 105-3022 bp; 3' half: NM_000350.2, bp 3023-6926) and MY07A was split between exons 24-25 (5' half: NM_000350.2, bp 273-3380; 3' half: NM_000350.2, bp 3381-6926). The ABCA4 and MYO7A proteins were both tagged at their C-terminus: ABCA4 with either the 3xflag (gactacaaagaccatgacggtgattataaagatcatgacatcgactacaaggatgacgatgacaag) or hemagglutinin (HA) tag (tatccgtatgatgtgccggattatgcg); MYO7A with the HA tag only. To generate trans-splicing and hybrid vector plasmids the CEP290 CDS was split at a natural exon-exon junction: between exons 29-30 (5' half: NM_025114, 345-3805; 3' half: NM_025114, 3806-7784). The CEP290 protein was tagged at its C-terminus with the hemagglutinin (HA) tag. The splice donor (SD) and splice acceptor (SA) signals contained in trans-splicing and hybrid dual AAV vector plasmids are as follows: 5'GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTT GTCGAGACAGAGAAGACTCTTGCGTTTCT-3' (SD) SEQ ID No. 1; 5'GATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG-3' (SA), SEQ ID No. 2. The recombinogenic sequence contained in hybrid AP vector plasmids (present in both first and second plasmids) were derived from alkaline phosphate (AP) genes (NM_001632, bp 823-1100), as previously described (39). The recombinogenic sequence contained in hybrid AK vector plasmids (present in both first and second plasmids) were derived from the phage F1 genome (Gene Bank accession number: J02448.1; bp 5850-5926).

[0066] The AK sequence is:

5'GGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAA TTTAACGCGAATTTTAACAAAAT-3', SEQ ID No. 3.

[0067] The ubiquitous CMV promoter is that contained in pZac2.1 (52) or pAAV2.1-CMV-EGFP (53); the ubiquitous CBA promoter was derived from pAAV2.1-CBA-EGFP (77), the PR-specific human RHO and RHOK promoters were derived from pAAV2.1-RHO-EGFP and pAAV2.1 RHOK-EGFP, respectively (70); the RPE-specific Vmd2 promoter (NG_009033.1, 4870-5470bp) corresponds to the previously described EcoRI-Xcml promoter fragment (47) and was amplified by human genomic DNA.

[0068] To generate dual AAV hybrid AK vectors with heterologous ITRs from AAV serotype 2 and 5 we exchanged the left ITR2 of the 5'-half plasmid and the right ITR2 of the 3'-half plasmid with the ITR5 (as depicted in Figure 17a). The plasmids for the production of AAV2 vectors with heterologous ITRs are the following: pZac5:2-CMV-5'A6CA4-SD-AK, pZac2:5-AK-SD-3'ABCA4-3xflag, pAAV5:2-CBA-5'/WYO7A-SD-AK and pAAV2:5-AK-SD-3'/WYO7AHA (Table 1).

Sequences [0069] ABCA4 gene

pZac2.1 -CMV-ASCA4_5'AK

Left ITR2 CTGCGGGCTCGCTGGGTGACTGAGGGGGCCGGGGGAAAGGCGGGGCGTCGGGGGACGTTTGG:

TCGCCCGGCCTCAGTGAGCGAGCGAGGGCGCAGAGAGGGAGTGGCGAAGTCGATCAGTAGGG GTTCCT (SEQ ID No. 4)

Left ITR5 CTCTCCCCGCTGTCGCGTTCGCTCGCTCGCTGGCTCGTTTGGGGGGGTGGCAGCTCAAAGAG·

CTGCCAGACGACGGCCCTCTGGCCGTCGCCCCCCCAAACGAGCCAGCGAGCGAGCGAACGCG ACAGGGGGGAGAGTGCCACACTCTCAAGCAAGGGGGTTTTGTAAGCAGTGA (SEQ ID No. 5) CMV enhancer

TCAATATTGGGCATTAGCCATATTATTCATTGGTTATATAGGAGAAATCAATATTGGCTATT

GGCGATTGCATACGTTGTATGTATATCATAATATGTACATTTATATTGGGTCATGTCeAATA TGACCGCCATGTTGGGATTGATTATTGAC (SEQ ID No. 6) CMV promoter tagttattaacagtaatcaattacggggtcattagttcatagcccatatatggagttccgcg

TTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACG tcaataatgacgtatgttggcatagtaacgccaatagggactttccattgacgtcaatgggt

GGAGTATTTACGGTAAACCGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGC cccctattgacgtcaatgacggtaaatggcccgcctggcattatgccgagtacatgacctta CGG G AC T TTC C T ACT T GGCAGT AGATCTACGTAT TAGTCATCGC TAT TACCATGGTGATGCΘ

GTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCC

ACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGT

CGTAATAAC C C C GCC C C GT T G ACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC TATAT AAG CAGAGC T CGT TTAG T GAACCGT (SEQ ID No. 7)

Chimeric intron

GT AAG T AT G AAGGTTAG AAG AG AGGTT T AAGGAG AGC-AAT AG AAAC T GGG G TTG T G GAG AG A

GAGAAGACTCT T GC GT TT GT GATAGGCACC T ATTGGT CTT ACT GACATC CAC TT T GC C T TT G TCTCCACAG (SEQ ID No. 8).

Abca4 5' atgggcttcgtgagacagatacagcttttgctctggaagaactggaccctgcggaaaaggca aaagattcgctttgtggtggaactcgtgtggcctttatctt-tatttctggtcttgatctggt

TAAGGAATGCCAACCCGCTCTACAGGCATGATGAATGCCATTTCGCCAACAAGGCGATGCGG

TCAGCAGGAATGCTGCCGTGGCTCCAGGGGATOTTCTGCAATGTGAACAATCCCTGTTTTCA

AAGCCCCACCCGAGGAGAATCTCCTGGAATTGTGTCAAACTATAACAACTCCATCTTGGCAA GGGTATATCGAGATTTTCAAGAACTCCTCATGAATGCACCAGAGAGCCAGCACCTTGGCCGT ATTTGGACAGAGCTACACATCTTGTCCCAATTGATGGACACGCTGGGGACTCACCCGGAGAG AATTGCAGGAAGAGGAATTGGAATAAGGGATATGTTGAAAGATGAAGAAAGACTGACACTAT tigτeatiaaaaacatcggcctgtct gact cagt gg tctacc t tctgatcaactctcaagt c cgtccagagcagttcgctcatggagtcccggacctggcgctgaaggacatcgcctgcagcga ggccctcgtggagcgcttcatcatcttcagccagagacgcggggcaaagacggigcgctatg CCCTGTGCTCCCTCTCCCAGGGCACCCTACAGTGGATAGAAGACACTCTGTAIGCCAACGTG GACTTCTTCAAGCTCTTCCGTGTGCTTCCCACACTCCTAGACAGCCGTTCTCAAGGTATCAA tgtgagatgttggggaggaatattatctgatatgtcacgaagaattgaagagtttatccatc GGCGGAGTATGCAGGACTTGCTGTGGGTGACCAGGeCGCTCATGCAGAATGGTGGTCCAGAG acctttacaaagctgatgggcatcctgtctgacctcctgtgtggctaccccgagggaggtgg CTGTCGGGT GCICTCCTIC AACTGGIATGAAGAC AATAACTATAAGGCCTITCIGGGGATIG ACTCCACAAGGAAGGATCCTATCTATTCTIAIGACAGAAGAAGAAGATCCTTIIGTAATGCA T T GATCCAGAGC C TGGAGT C AAATCCT T T AACCAAAATCGCITGGAGGGCGGCAAAGCCIT T GCTGATGGGAAAAATCCTGTACACTCCTGATTCACCTGCAGCACGAAGGATACTGAAGAATG CCAAC T CAACT T TT GAAGAACTGGAACACGTTAGGAAGTT GGT CAAAGCC T GGGAAGAAGTA gggccccagatctggtacttcttigacaacagcacacagatgaacatgatcagagataccct ggggaacccaacagtaaaagactttttgaataggcagcttggtgaagaaggtaitactgctg aagccatcctaaacttcctciacaagggccctggggaaagccaggctgacgacatggccaac ttggactggagggacatatttaacatgactgatcgcaccctccgccttgtcaaicaatacct GGAGT GC TTGGTCCTGGATAAGTT TGAAAGC T AC AATGATGAAACTCAGC T CACCCAAC GTG CCCTCTCTCTACTGGAGGAAAACATGTTCTGGGCCGGAGTGGTATTCCCTGACATGTATCCC iggaccagctctctaccaccccacgigaagtataagaiccgaaiggacaiagacgtggtgga gaaaaccaataagattaaagacaggiattgggattctggtcccagagctgatcccgtggaag atttccggtacatctggggcgggtttgcctatctgcaggacatggttgaacaggggatcaca aggagccaggtgcaggcggaggctccagttggaatctacctccagcagatgccctacccctg cttcgtggacgattctttcatgatcatcctgaaccgctgtttccctaicttcaiggtgcigg CATGGAT CTAC T CT GT CICCATGACIGTGAAGAGCATCGT CTTGGAGAAGGAGTT GCGACT G AAGGAGACCTTGAAAAATCAGGGTGTClCCAAIGCAGTGATTTGGTGIACCTGGTieCTGGA CAGC TTCI G C AT CATGTCGAT GAGCATCTTCCICCTG ACGAT AT TEATCATGCAT GGAAGAA TCCTACATTACAGCGACCCATTCATCCTCTTCCTGTTCTTGTTGGCTTTCTCCACTGCCACe ATCATGCTGTGCTTTCTGCTCAGCACCTTCTTCTCCAAGGCCAGTCTGGCAGCAGCCTGTAG TGGTGTCATCTATTTCACCCTCTACCTGCCACACATCCTGTGCTTCGCCTGGCAGGACCGCA TGACCGCTGAGCTGAAGAAGGCTGTGAGCTTATTGTCTCEGGIGGCATTTGGATTTGGCACI gagtacciggttcgctttgaagagcaaggcctggggctgcagtggagcaacatcgggaacag TCCCACGGAAGGGGACGAATTCAGCTTCCTGCTGiecATGCAGATGATGCTCCTTGATGCTG ctgtctatggcttactcgcttggtaccttgatcaggtgtttccaggagactatggaacccca CTTCCTTGGTACTTTCTTCTACAAGAGTCGTATTGGCTTGGCGGTGAAGGGTGTTCAACeAG agaagaaagaggcctggaaaagaccgagcccciaacagaggaaacggaggatccagagcacc CAGAAGGAATACACGACTCCIICITTGAACGTGAGCATCCAGGGTGGGTICCTGGGGTATGC GTGAAGAATCTGGTAAAGATTTTTGAGCCCTGTGGCCGGeCAGCTGTGGACCGTCTGAAeAT CACCTTCIACGAGAACCAGATCAeCGCATTCCTGGGCCACAATGGAGCTGGGAAAACCACCA CCTT (SEQ ID No. 9)

Splicing donor signal GTAAGTATGAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACA GAGAAGACTCTTGCGTTTCT (SEQ ID No. 1)

AK gggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgc GAATTTTAACAAAAT (SEQ ID No. 3)

Right ITR2 (or 5' ITR2) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCC GGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGC GCGCAG (SEQ ID No. 10)

Full length sequence of pZac2.1-CMV-ASCA4_5'AK

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGG

TCGCCGGGGGTCAGTGAGCGAGGGAGGGGGGAGAGAGGGAGTGGCCAACTGCATCACTAGGG

GTTCCTTGTAGTTAATGATTAACCCGGCATGCTACTTATCTACGTAGCCATGCTCTAGGAAG

ATCITCAATATTGGCCAITAGCCATAITATTCAIIGGTTATATAGCAJIAAAICAATATTGGC

TATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTGATGTCC

AATATGAGGGCCATGTTGGGATTGATTATTGACTAGTTATTAATAGTAATGAATTAGGGGGT cattagttcatagcccatatatggagttccgggttacataacttacggtaaatggcgcgcct

GGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAAC

GGGAATAGGGACTTTGCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCGCACTTGG gagtagatcaagtgtatcatatggcaagtcgggcccctattgacgtcaatgacggtaaatgg

CCGGCGTGGGATTATGCGCAGTACATGAGGTTACGGGAGTTTCCTACTTGGGAGTACATGTA cgtattagtcatcgctattaccatggtgatgcggttttggcagtacaccaatgggcgtggat

AGCGGTTTGACTCACGGGGATTTCGAAGTCTCGACCCCATTGACGTGAATGGGAGTTTGTTT TGGCACCAAAATCAACGGGACΤTTCCAAAATGTCGTAATAACCCCGCCCGGTTGACGCAAAT gggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcaga ΪCACTAGAAGCITTATT GCGGIAGT IT AT CAC AGTTAAAT TGCTAACGCAG TCAGT GC ITC T GACACAACAGACTCGAACTTAAGCTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTAT CAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACCGGGCTCGTCGAGACAGAGAAGAC TCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTIGCCITICTCTCCACA GGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTCACTATA GGCTAGCCTCGAGAATTCACGCGTGGTACCTCTAGAGTCGACCCGGGCGGCCGCCATGGGCT TCGTGAGACAGATACAGCTTTTGCTCTGGAAGAACTGGACCCIGCGGAAAAGGCAAAAGATT CGCTTTGTGGIGGAACTCGTGIGGCCTTTATCTTTATICCTGGTCTIGATCTGGTTAAGGAA T GC CAAC CC GCT C TACAGCCATCATGAATGCCATTITCCCAACAAGGCGAIGCCCTCAGCAG GAATGCTGCCGTGGCTCCAGGGGATCTTCIGCAATGTGAACAATCCCTGTTTTCAAAGCCCC ACCCCAGGAGAATCTCCTGGAATTGTGTCAAACTATAACAACTCGATCTTGGGAAGGGTATA TCGAGATTTTGAAGAACTCCTCAT GAATGCAC CAGAGAGCCAGCACCIT GGCCGTATII GG A CAGAGCTACACATCTTGTCCCAATTCATGGACACCCTCeGGACTCACCCGGAGAGAATTGCA GGAAGAGGAATTCGAATAAGGGATATCTTGAAAGATGAAGAAACACTGACACTATTTCTCAT taaaaacatcggcctgtctgac.tcagtggtctaccttccgatcaactctcaagtccgtccag agcagttcgctcatggagtcccggacctggcgctgaaggacatcgcctggagggaggccctc ctggagcgcttcatcatcttcagccagagacgcggggcaaagacggtgcgctatgccctgtg

CTCCCTCTCCCAGGGCACCCTACAGTGGATAGAAGACACTCTGTATGCCAACGTGGACTTeT TCAAGCTCTTCCGTGTGCTTCCCACACTeCTAGACAGCCGTTCTCAAGGTATCAATCTGAGA T CTT GGGGAGGAATAT TATGT GATATGTCACCAAGAATT CAAGAG T ITATCCATTGGCC GAG TATGCAGGACTTGCTGTGGGTGACCAGGCC'CCTCATGCAGAATGGTGGTCCAGAGACCTTTA eAAAGGTGATGGGCATCCTGTCTGACCTCCTGTGTGGCTAeCCeGAGGGAC-GTGGCTGTCGG GTGCTGTCCTTGAACTGGTATGAAGACAATAACTATAAGGCCTITCTGGGGATTGACTCCAC AAGGAAGGATCCTATCTATTCTTATGACAGAAGAACAACATCCTTTTGTAATGCATTGATCC· AGAGCCTGGAGTCAAAT CCTITAACCAAAATCGCTTGGAGGGCGGCAAAGGCITTGC TGATG: GGAAAAAICCTSTACACTCCTGATTCACCTGCAGCACGAAGGATACTGAAGAATGCCAACIC AACTTTTGAAGAACTGGAACAeGTTAGGAAGTTGGTCAAAGCCTGGGAAGAAGTAGGGCeCC AGATCTGGTACTTCIITGACAACAGCACACAGATGAACATGATCAGAGATACCCTGGGGAAC CCAACAGTAAAAGACTTTTTGAATAGGCAGCTTGGTGAAGAAGGTATIACTGCTGAAGCCAT CCTAAAGTTCCTCTACAAGGGCCCIGGGGAAAGCCAGGCTGACGACATGGCCAACTTCGACT GGAGGGACATATTTAAeATCACTGATeGCACCCTCCGeCTTGTCAATeAATACCTGGAGTGC ITGGICCIGGATAAGTTIGAAAGCIACAATGAIGAAACCCAGCieACCeAACGIGCCCTCTC tctactggaggaaaacatgttctgggccggagtggtattccctgacatgtatccctggacca GC Τ C T C TAC CACCCCACG T GAAG TATAAGATCC GAATGGACATAGACS T GG TGGAGAAAAC C AATAAGATIAAAGACAGGIATTGGGATTCTGGTCCeAGAGCTGATCCCGTC-GAAGATTTCCG GIACATCTGGGGCGGGTTTGCCTATCTGCAGGACATGGTTGAACAGGGGATCACAAGGAGCC AGGTGCAGGCGGAGGeTCCAGTTGGAATCTAeCTCCAGCAGATGCCeTACCCCIGCTICGIG GACGATTCTTTCATGATCATeCTGAACCGCTGTTTCCCTATCTTCATGGTGCTGGCATGGAT CTACTCTGTCTCCATGACTGTGAAGAGCATCGTCTTGGAGAAGGAGTTGCGACTGAAGGAGA CCTTGAAAAATGAGGGTGTCTCCAATGCAGTGATTTGGnGTACCTGGTTCCTGGACAGCTTC tccatcatgtcgatgagcatcttcCtcctgacgatattcatcatgcatggaagaatcctaca TTACAGCGACCCATTCAICCTCTTCCIGTTCTTGTTGGCTTTCTCCACTGCCACCATCATGC IGIGCTTTCTGClCAGCACCTTCTTCTCCAAGGCCAGCCTGGCAGCAGCCIGIAGTGGTGIC tgaGctgaagAaggctgtgagcttactgtctccggtggcatttggatttgggactgagtacc

TGGTTCGGTTTGAAGAGCAAGGCCTGGGGGTGGAGTGGAGCAAGATGGGGAAGAGTCCCACG

GAAGGGGACGAATTCAGCTTCC GGCIGTCC ATGCAGATGAT GCT CC T T GATGC TGCTGT CTA

TGGCTTACTCGCTTGGTACCTTGATCAGGTGTTTCCAGGAGACTATGGAACCCCACTTCCTT

GGTACTT T GT TGTACAAGAGT C GTAT TGGCTT GGCGGT GAAGGGTG T T GAACCAGAGAAGAA

AGAGCCCTGGAAAAGACCGAGCCCCTAACAGAGGAAACGGAGGATCCAGAGCACCCAGAAGG

AATACACGACTCCTTCTTTGAACGTGAGCATCCAGGGTGGGTTCCTGGGGTATGCGTGAAGA

ATCTGGTAAAGATTTTTGAGCCCTGTGGCCGGCCAGCTGTGGACCGTCTGAACATCACCTTC

TACGAGAACCAGATCACCGCATTCCTGGGCCACAATGGAGCTGGGAAAACCACCACCTTGTA

AGTAT CAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTT GT CGAGACAGAG

AAGACTCTTGCGTTTCTGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATT

TAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTATAATTTCAGGTGGCATCTT

T CCAATTGAGGAACCCC TAGTGAT GGAGTT GGCCAC TCCC T CTC TGCGCGC T CGC T CGCTCA ctGaggccgGgcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagc GAGCGAGCGCGCAG (SEQ ID No. 11) pZac2.1 -ASCA4_3'AK_SV40

Left ITR2

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGG

TCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGG GTTCCT (SEQ ID No. 4)

AK

GGGAT T T TGC C GATTT C GGC CTATT GGT TAAAAAATΘAGC TGAT TTAACAAAAAT T TAAC GC GAATTTTAACAAAAT (SEQ ID No. 3)

Splicing acceptor signal GATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG (SEQ ID No.2)

Abca4_3'

GTCCATc€TGACGGGTCTGTTGCCACCAACCTCTGGGACTGTGCTCGTTGGGGGAAGGGACA

TTGAAACCAGCCTGGATGCAGTCCGGCAGAGCCTTGGCATGTθTCCACAGCACAACATCCTG

TTCCACCACCTCACGGTGGCTGAGCACATGCTGTTCTATGCCCAGCTGAAAGGAAAGTCGCA

GGAGGAGGCCCAGCTGGAGATGGAAGCCATGTTGGAGGACACAGGCCTCCACCACAAGCGGA

ATGAAGAGGCTCAGGACCTATCAGGTGGCATGCAGAGAAAGCTGTCGGTTGCCATTGCCTTT

GTGGGAGATGCCAAGGTGGTGATTCTGGACGAACCCACCTCTGGGGTGGACCCTTACTCGAG acgctcaatctgggatctgctcctgaagtatcgc.tcaggcagaaccatcatcatgtccactc accacatggacgagGccgacctccttggggaccgcattgccatcattgcccagggaaggctc

TACTGCTCAGGCACCCCACTCTTCCTGAAGAACTGCTTTGGCACAGGCTTGTACTTAACCTT

GGT GC GCAAGAT GAAAAACATCCAGAGCCAAAGGAAAGGCAGTGAGGGGACCTGC AGC T GC T

CG TCTAAGGGTTTCTCCACCACGTGT CCAGCCCACGTCGATGACCTAACT CCAGAACAAGT C

CTGGATGGGGATGTAAATGAGCTGATGGATGTAGTTCTCCACCATGTTCCAGAGGCAAAGCT

GGTGGAGTGCATTGGTCAAGAACTTATGTTCCTTCTTCCAAATAAGAACTTCAAGCACAGAG

CATATGCCAGCCTTTTCAGAGAGCTGGAGGAGACGCTGGCTGACCTTGGTCTCAGCAGTTTT

GGAATTTCTGACACTCCCCTGGAAGAGATTTTTCTGAAGGTCACGGAGGATTCTGATTCAGG

ACCTCTGTTTGCGGGTGGCGCTCAGCAGAAAAGAGAAAACGTCAACCCCCGACACCCCTGCT

TGGGTGCCAGAGAGAAGGCTGGACAGACACCeCAGGACTGCAATGTCTGCTCCCCAGGGGCG

CCGGCTGCTCACCCAGAGGGCCAGCCTCCCCCAGAGCCAGAGTGCCCAGGCCCGCAGeTCAA cacggggacacagctggtcctccagcatgtgcaggcgctgctggtcaagagattccaacaca ccatccgcagc cacaaggact t cct ggcgcagat cgtgctccc ggctacctt tgt gt t TT t g gctctgatgctttctattgttatccctccttttggcgaataccccgctttgacccttcaccc ctggatatatgggcagcagtacaccttcttcagcatggatgaaccaggcagtgagcAgttca cggtactigcagacgicctcgtgaataaggcaggctttggcaaccgctgcctgaaggaaggg

TGGCTTCCGGAGTACCCCTGTGGCAACTCAACAGCCTGGAAGACICCTTCTGTGTCCCCAAA CATCACCCAGCTGTTCCAGAAGCAGAAATGGAGACAGGTCAACCCTTCACCATCCTGCAGGT gcagcaccagggagaagctcaccatgctgccagagtgccccgagggtgccgggggcctcccg CCCCGCCAGAGAACACAGCGCAGCACGGAAATICTACAAGACGTGACGGACAGGAACATCTG CG AC TTCTTGGTAAAAACG TAT CCTGC T CT TATAAGAAGCAGCITAAAGAGCAAAT T CTGGG TCAATGAACAGAGGTATGGAGGAATTTCCATTGGAGGAAAGCTCCCAGTCGTCCCCATCACG GGGGAAGCACTTGTTGGGTTTTTAAGGGACCTTGGCCGGATCATGAATGTGAGCGGGGGCCC TATCACTAGAGAGGCCTCTAAAGAAATACCTGATTTCCTTAAACATCTAGAAACTGAAGACA ACATTAAGGTGTGGTTTAATAACAAAGGCTGGCATGCCCTGGTCAGCTTTCTCAATGTGGCC CACAACGCCATCTTACGGGCCAGCCTGCCTAAGGACAGAAGCCCCGAGGAGTAIGGAATCAC GGT CAT TAGCCAACCCC TGAAGC T GACCAAG GAGCAG C TC T CAGAGAT TACAGTGGTG AC C A CTTCAGTGGATGCTGTGGTTGCCATCTGCGTGATTTTCTCCATGTCCTTCGTCCCAGCCAGC TTTGTCCTTTATTTGATCCAGGAGCGGGTGAACAAATeCAAGCACCTCCAGTTTATCAGTGG AGCGAGGGGCACCAeCIACTGGGTAACCAACTTCCTCTGGGACATCATGAATTAITCCGTGA G T G C T GGGCTGGTGGTGGGCATC11CAT CGGGTT TCAGAAGAAAGCCTACACTTCIC CAGAA AAC CT TCCTGCCC TT GT GGCAC T G C TCCTGC TGTAT GGAT GGGCGGT CAT T CCC AT GAT GTA CCCAGCAT CCT T CCT GT T T G ATG T CGG CAGCACAGCCTATGCGGGTTTATCT TG T GC TAAT C TGTTCATCGGCATCAACAGCAGTGCTATTACCTTCATCTTGGAATTATTTGAGAATAACCGG ACGCTGCTCAGGTTCAACGCCGTGCTGAGGAAGCTGCTCATTGTCTTCCCCCACTTCTGCCT gggccggggcctcattgaccttgcactgagccaggctgtgacagatgtctatgcccggtttg gtgaggagcactctgcaaatccgttccactgggacctgattgggaagaacctgtttgccaig gtggtggaaggggtggtgtacttcctcctgaccctgctggtccagcgccacttcttcctcic eCAATGGATCGCCGAGCeGACIAAGGAGCCCATTGTTGATGAAGATGATGATGTGGCTGAAG AAAG ACAAAGAATTAT TAG T GGT GGAAATAAAAC T GACAT CT TAAGGC TACATGAACTAACC AAGATTTATCCAGGCACCTCCAGCCCAGCAGTGGACAGGCTGTGTGTGGGAGTTCGCCCTGG AGAGTGCTTTGGCCTCCTGGGAGTGAATGGTGCCGGCAAAACAACCACATTCAAGATGCTCA CTGGGGACACCACAGTGACCTCAGGGGATGCCACCGTAGCAGGCAAGAGTATTTTAACCAAT ATTTCTGAAGTCCATCAAAATATGGGGTACTGCCCTCAGTTTGATGCAATCGATGAGCTGCT cacaggacgagaacatctttacctttatgcccggcttcgaggtgtaccagcagaagaaatcg AAAAGGTTGCAAACTGGAGTATTAAGAGCCTGGGCCTGACTGTCTACGCCGACTGCCTGGCT GGCACGTACAGTGGGGGCAACAAGCGGAAACCeiCCACAGCCATCGCACTCATTGGCTGCCC accgctggtgctgctggatgagccCaccacagggatggacccccaggcacgccgcatgctgt ggaacgtcatcgtgagcatcatcagagaagggagggctgtggtcctcacatcccacagcatg GAAGAAT GTGAGGCAGTGTGTACCCGGCTGGCCATCAT GGTAAAGGGCGCC Τ Τ T C GATGTAT GGGCACCATTCAGCATCTCAAGTCCAAAITIGGAGATGGCTAIATCGTCACAATGAAGATCA AAT CC CC GAAGGACGACC TGC T TCCTGACC ΤGAACCCTGT GGAGCAGT TGT T CCAGGGGAAC TTCCeAGGCAGTGTGCAGAGGGAGAGGCACTACAACATGCTCCAGTTCCAGGTCTCCTCCTC CTCCCTGGCGAGGATCTTCCAGCTCCTCCTCTCCCACAAGGACAGCCTGCTCATCGAGGAGT ACT CAGT CACACAGAC CACAC T GGAC CAGGT GT T TGTAAAT TT TGCIAAACAGCAGACT GAA AGTCATGACCTCCCTCTGCACCCTCGAGCTGCTGGAGCCAGTCGACAAGCCCAGGACGACTA CAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGT GAGCGGCCGC (SEQ ID No. 12)

Sv40 polyA Τ T C GAGCAQACAT GATAAGATACAT T GATGACTTTGGACAAACCACAAC TAGAATGCAGTGA aaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctg CAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGT GGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGACCTieCT AGAGCATGGCTAC (SEQ ID No. 13)

Right ITR2 AGGAACCCCTAGTGAIGGAGTTGGCCACTCCCTCTCTGCGCGCICGCTCGCICACTGAGGCC GGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGC GCGCAG (SEQ ID No. 10) RIGHT ITR5 tcactgcttacaaaacgcccttgcttgagagtgtggcactctcccccctgtcgcgttcgctc GCTCGCTGGCTCGTTTGGGGGGGCGACGGGCAGAGGGCCGTCGTCTGGCAGCTCTTTGAGCT GCCACCCCCCCAAACGAGCCAGCGAGCGAGCGAACGCGACAGGGGGGAGAG (SEQ ID No. 141 -*· /

Full lenght sequence of pZac2.1-ASCA4_3'AK_SV40 CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGG TCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGG GTTCCTGGATCCGGGATTTIGCCGATTTCGGCCIATTGGTTAAAAAAIGAGCIGATTIAACA AAAATTTAACGCGAATTTTAACAAAATATTAACGITTATAATT TCAGGTGGCATCT T TCGAT AGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTCCATCCTGACGG G TC TG T TGC CACCAACC TC TGGGAC TGT GCICGTTGGGGGAAGGGACATTGAAACCAGC CTG GATGCAGTeCGGCAGAGCCTTGGCATGTGTCCACAGCACAACATCCTGTTCCACCACCTCAC GGTGGCTGAGCACATGCTGTTCTATGCCCAGCTGAAAGGAAAGTCCCAGGAGGAGGCCCAGC TGGAGATGGAAGCCATGTTGGAGGACACAGGCCTCCACCACAAGCGGAATGAAGAGGeTCAG GACCTATCAGGTGGCATGCAGAGAAAGCTGTCGGTTGCCATTGCCTTTGTGGGAGATGCCAA GGTGGT GAT T CT GGACGAAC CCACCT C TGGGGTGGAC CC Τ TACT CGAGACGCTCAATCT GGG ATCTGCTCCTGAAGTATCGCTCAGGCAGAACCATCATCATGTCCACTCACCACATGGACGAG GCCGACCTGCTTGGGGACCGCATTGCCATCATTGCCCAGGGAAGGCTCTACTGCTCAGGCAC CCCACTCTTCCIGAAGAACTGCTTTGGCACAGGCTIGIACTTAACCTTGGTGCGCAAGATGA aaaacatccagagccaaaggaaaggcagtgaggggacctgcagcigctcgtgtaagggtttc tccaccacgtgtcgaggccacgtcgatgacctaactccagaacaagtcctggatggggatgt AAATGAGCTGATGGATGTAGTΤ CTCCACCATGTTCCAGAGGCAAAGCTGGTGGAGTGCAT TG GTCAAGAAC T TATCT TCCT I CTT CCAAATAAGAAC TTCAAGCACAGAGCATATGCCAG GCΤ T TTCAGAGAGCTGGAGGAGACGCTGGCTGACCTTGGTCTCAGCAGTTTTGGAATTTCTGACAC TCCCCIGGAAGAGATΤ Τ T TCTGAAGGTCACGGAGGATTCTGAT TCAGGACCICIGTTTGCGG GTGGCGCTCAGCAGAAAAGAGAAAACGTCAACCCCCGACACCCCTGCΤTGGGTCCCAGAGAG AAGGCTGGACAGACACCCCAGGACTCCAATGTCTGCTCCCCAGGGGCGCCGGCTGCTCACCC agagggccagcctcccccagagccagagtgcccaggcccgcagctcaacacggggacacagc TGGTGCTCGAGCATGTGCAGGCGCTGCTGGTCAAGAGATTCCAACACACCATCCGCAGCCAC AAGG AC TI CCT GGCGC AGATCGTGC TCCC GGCTACCITTGT GT Τ Τ Τ TGGC T CIGAT GC Τ Τ T C tatigttatcccicgttttggcgaatacggggctttgagcgttgacccctggatatatgggg AGCAGTACACCTTCTTCAGCATGGATGAACCAGGCAGTGAGCAGTTCACGGTACTTGGAGAC GTCCTCCTGAATAAGCCAGGCTTTGGCAACCGCTGCCTGAAGGAAGGGTGGCTTCCGGAGIA CCCCTGTGGCAACTCAACACCCTGGAAGACTCCTTCTGTGTCCCCAAACATCACCCAGCTGT ICCAGAAGCAGAAATGGACACAGGTCAACCCTTCACCATCCTGCAGGTGCAGCACCAGGGAG AAGCTCACCATGCTGCCAGAGTGCCCCGAGGGTGCCGGGGGCCTCCCGCCCCCCCAGAGAAC ACAGC GCAGCAC GGAAAIICTACAAGACCTGACGGACAGGAACATC TC C GAC Τ T C Τ T GGTAA aaacgtatcctgctcttataagaagcagcttaaagagcaaattctgggtcaatgaacagagg TATGGAGGAATTTCCATTGGAGGAAAGCTCCCAGTCGTCCCCATCACGGGGGAAGCACTTGT tgggtttttaagcgacCttggccggatcatgaatgtgagcgggggccctatcactagagagg cctctaaagaaatacctgatttccttaaacatctagaaaatgaagacaacattaaggtgtgg T TTAATAACAAAGGCIGGCATGCCC T GGTCAGCT TT C T CAAT GTGGCCCACAACGCCATCTT ACGGGCCAGCCIGCCCAAGGACAGAAGCCGCGAGGAGTAIGGAATCACCGTCAITAGCCAAe

CCCTGAACCTGACCAAGGAGCAGCTCTCAGAGATIACAGIGCTGACCACTICAGTGGAIGCT GTGGTTGCgaTCTGCGTGAITTTCTCCATGICCTICGTCCCAGCCAGCTTTGTCCTITATIT GATCCAGGAGCGGGTGAACAAATCCAAGCACCTC CAGTΤTATCAGTGGAGTGAGCCCCAGCA CCTACTGGGTAACCAACTTCCICTGGGACATCATGAAT TATICCGT GAGTGCTGGGCTGGTG GT GGGCAT C T TCATCGGGΤΤIGAGAAGAAAGCCTACACITCICCAGAAAACC TICC T GC CC T TG'TGGCACTGCTCCTGCTGTATGGATGGGCGGTCATTCCCATGATGTACCCAGCATCCTTCC TGTTTGATGTCCCCAGCACAGCCIATGTGGCTTTATCTTGTGCTAATCTGTTCATCGGCATC AACAGCAGTGC TAT TACCTT CATCT T GGAAT TAT TIGAGAATAACCGGACGCTGCT C AGGT I CAACGCCGTGCTGAGGAAGCTGCTCATTGTCTTGCCCCACTTCTGCCTGGGCCGGGGCCTCA T TGAC CT T GCAC TGAGGCAGGCTGTGACAGAT GT CT AT GCCCGGTT TGGT GAGGAGCACTCT gcaaatccgttccactgggacctgattgggaagaacctgtttgccatggtggtggaaggggt GGTGTACTTCCTCCIGACCCTGCTGGICCAGCGCCACTTCTTCCTCTCCCAATGGATTGCCG agcccactaaggagcccattgttgatgaagatgatgatgtggctgaagaaagacaaagaatt ATIACTGGTGGAAATAAAACTGACATCTTAAGGCTAGATGAACTAACCAAGATΤTATCCAGG CACCTCCAGCCCAGCAGTGGACAGGCIGIGIGTCGGAGTTCGCCCTGGAGAGTGCTTTGGCC TCC T GGGAGT GAAT GGTGCC GGCAAAACAACCACAT TCAAGAT GCTCAC T GGGGACACCACA GTGACCTCAGGGGATGCCACCGTAGCAGGCAAGAGTAITTTAACCAATAITTCTGAAGTCCA ΤCAAAATATGGGCTACTGTCCTCAGTTTGATGCAATCGATGAGCTGCTCACAGGACGAGAAC ATCTTTACCTTTATGCCCGGCTTCGAGGTGTACCAGCAGAAGAAATCGAAAAGGTTGCAAAC tggagtattaagagcctgggcctgactgtctacgccgactgcctggctggcacgtacagtgg gggcaajcaagcggaaagtctccacagccatcgcactcattggctgcccaccgctggtgctgc tggatgagcccaccacagggatggacccccaggcacgccgcatgctgtggaacgtcatcgtg agcatcatcagagaagggagggctgtggtcctcacatcccacagcatggaagaatgtgaggc actgtgtacccggctggccatcatggtaaagggcgcctttcgatgtatgggcaccattcagc ATC T CAAGTCCAAAT TTGGAGATGGC TATATCGT CACAATGAAGATCAAATCCCCGAAGGAC GACCTGCTTCCTGACCTGAACCCTGTGGAGCAGTTCTTCCAGGGGAACTTCCCAGGCAGTGT GCAGAGGGAGAGGCACTACAACATGCTCCAGTICCAGGTCTCCTCCTCCTCCCTGGCGAGGA Τ CT τ CCAGC τ Cc TCC TCTGC CACAAGGACAGCCTGCTCATCGAGGAGTACT CAGT CACACAG accacactggaccaggtgtttgtaaattttgctaaacagcagactgaaagtcatgacctccc tctgcaccctcgagctgctggagccagtcgacaagcccaggactgagcgggcgcttcgagca gacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatg ctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaac aagttaacaacaacaattgcattcattttatgtttcaggttcagggggagatgtgggaggtt TTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGATCTTCCTAGAGeATG GCTACGTAGATAAGTAGGATGGCGGGTTAATCATTAACTACAAGGAACCGCTAGTGATGGAG TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCG ACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG (SEQ ID No . 15)

pZac2.1 -CMV-ABCA4_5’TS

Full length sequence of pZac2.1-CMV-ASCA4_5'TS

ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcCcgggcgtcgggCgacctttgg tcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggg gttcgttgtagttaatgattaacccgccatgctacttatctacgtagccatgctctaggaag ATC ΤTCAATATTGGCCATIAGCCATATTATTCAT TGGTTATATAGCATAAAT CAAT ATT GGC TATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCC AATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGT CATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCeT GGC T GACC GCCC AACGACC CCCGCCCATTGACGTCAATAATGACGTAT GTTCCCATACTAAC

gccaatagggactttccatcgacgtcaacgggtggagtattiacggtaaactgcccacttgg; CAGTACATCAAGTGTATCATATGCCAAGTCCGCCCeeTATTGACGTCAATGACGGTAAATGG cccgcctggcattatgcccagtacatgaccttacgggactttcctacttgccagtacatcta CGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGAT AGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTT tggcaccaaaatcaacgggactttccaaaatgtcgtaacaaccccgcccccttgacgcaaat GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGA tcactagaagctitatigcggtagtttatcacagttaaattgctaacgcagtcagtgcttct gacacaacagtctcgaacttaagctgcagaagttggtcgtgaggcactgggcaggtaagtat CAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGAC tcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccaca GGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTCACTATA GGC-TAGCCTCGAGAATTCACGCGTGGTACCTCTAGAGTCGACCCGGGCGGCCGCCATGGGCT TCGTGAGACAGATACAGCTTTTGCTCTGGAAGAACTGGACCCTGCGGAAAAGGCAAAAGATT CGCTTTGTGGTGGAACTCGTGTGGCCTTTATCTTTATTCCTGGTCTTGATCTGGTTAAGGAA TGCCAACCeGCTCTACAGCCATCATCAATGCCATTTCCCCAACAAGGCGATGCCCTCAGCAG GAATGCTGCCGTGGCTCCAGGGGATCTTCTGCAATGTGAACAATCCCTGTTTTCAAAGCCCC ACeeCAGGAGAATCTCCTGGAATTGTGTCAAACTATAACAACTCCATCTTC-GeAAGGGTATA tcgagaTttTcaagaactcctcatgaatgcaccagagagccAgcaccttggccgtatttgGa CAGAGCT ACAC A T C T TGTCCC AATTCATG GAG ACCCT CC GGAC TC AC C C GG AGA GAAT T GCA ggaagaggaattcgaataagggatatcttgaaagatgaagaaacactgacactatttctcat TAAAAACAT C G GC C T GTCTGACT CAGTGGTCTACCT TC TGATCAACICTCAAGTCCGTCCAG agcagttcgctcatggagtcccggacctggcgctgaaggacatcgcctgcagcgaggccctc CTGGAGCGCTTCATCATCTTCAGCCAGAGACGCGGGGCAAAGACGGTGCGCTATGCCCTGTG: CTCCCTCTCCeAGGGeACCCTACAGTGGATAGAAGACACTCTGTATGCCAACGTGGACTTCT TC AAGCTC Τ TCGGTGTGCT TCCCACAC TCC TAGACAGC CGT TCTCAAGGTAT CAATC T GAGA TCTTGGGGAGGAATATTATCTGATATGTCACCAAGAATCCAAGAGTTTATCCATCGGGOGAG TATGCAGGACTTGCTGTGGGTGACCAGGCCCCTCATGCAGAATGGTGGTGCAGAGAC.CTTTA

CAAAGC T GAT GGG CATC CTGTCIGACCTCC T GTGTGGCTACCCCGAGGGAGGT GGC T C T CG G; GTGCT'CTCCTTCAACTCGTATGAAGACAATAACTATAAGGCCTTTCTGGGCATTGACTCCAC' AAGGAAGGATCCTATCTATTCTTATGACAGAAGAACAACATCCTTTTGTAATGCATTGATCC AGAGCCTGGAGTCAAATCCTTTAACCAAAATCGCTTGGAGGGCGGCAAAGCCTTTGCTGATG GGAAAAATCCTGTAGACTCCIGATTCACCTGCAGCACGAAGGATACTGAAGAATGCCAACTC AACTITTGAAGAACT GGAACACGΤTAGGAAGΤ T GGTCAAAGCCTGGGAAGAAGTAGGGCCCC AGATCTGGT ACT TC IΤ T GACAAC AGCACACAGATGAAC ATGAT C AGAC-AT ACCC TGGGGAAC CCAACAGTAAAAGACTTTTTGAATAGGCAGCTTGGTGAAGAAGGTATTACTGCTGAAGCCAT cctaaacttcctctacaagggccctcgggaaagcgaggctgacgacatggccaagttcgact ggagggacatatttaacatcactgatcGcaccCtccgccttgtcaatCaataCctggaGtgc ttggtcctggataagtttgaaagctaCaatgatgAaactcaGctCacccaacgtgccctctc tctactggaggaaaacatgttctgggccggagtggtatcccctgacatgtatccctggacca gctccctagcaccccacgtgaagtataagatgcgaatggacaiagacgtggtggagaaaacc aataagatiaaagacaggtaitgggaticiggtcgcagagcigatccogtggaagattiggg gtacatctggggcgggtttgcctaicigcaggacatggctgaacaggggatcacaaggagcc AGGTGCAGGCGGAGGeTCCAGTTGGAATCTACCTCCAGCAGATGCCCTACCCCTGCTTCGTG gacgattctttcatgatcatcctgaaccgctgtttccctatcttcatggtgctggcatggat CTACTCTGTCTCeATGACTGTGAAGAGCATCGTCTTGGAGAAGGAGTTGCGACTGAAGGAGA ΤΰΤΤ6ΛΑΑΑΑΤΤΑΤβθΙ6ΤϋΤΤΤΑΑΤΘΤΑΘΤ6ΑΤΤΤΘΘπ6ΤΑθσΤΤΤΤΤΤΟΤΘΟΑΰΑΘΤΤΤΤ TCCATCATGTCGAT GAGC AT CIICC TCC TGACGATAT T CATCATGCAT GGAAGAATCC TAGA ttacagcgacccattcatcctcttcccgttcttgttggctttctccactgccaccatcatgc TGTGCTTTCTGCTCAGCACCTTCTTCTCCAAGGCCAGTCTGGCAGCAGCCTGTAGTGGTGTC ATCTATT ICACCCTCTACCIGCCAGACATCCIGTGC ΤICGCCTGGCAGGACCGCATGACCGC TGAGCTGAAGAAGGCTGTGAGCTTACTGICTCCGGTGGCATTTGGATTTGGCACTGAGTACC IGGTTCGCCTTGAAGAGCAAGGCCTGGGGCTGCAGIGGAGCAACATCGGGAACAGTCCCAGG GAAGGGGACGAATTCAGCTTCCTGCTGTCCATGCAGATGATGCTCCTTGATGCTGCTGTCIA TGGCTTACTCGCTTGGTACCTTGATCAGGTGTTTCCAGGAGACTATGGAACCCCACTTCCTT GG TAC Τ Τ T C T TC TAGAAGAGT CGTAT TGGCTTGGCGGT GAAGGGTGTTCAACCAGAGAAGAA. agagccciggaaaagaccgagcccctaacagaggaaacggaggaiccagagcacccagaagg AATACACGACTCCTTCTTIGAACGTGAGCATCCAGGGTGGGTTCCTGGGGTATGCGTGAAGA ATCTGGTAAAGATTTTTGAGCCCTGTGGCCGGCCAGCTGTGGACCGTCTGAACATCACCTTC TACGAGAACCAGATCACCGCATTCCTGGGCCACAATGGAGCTGGGAAAACCACCACCTTGTA AGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAG aagactcttgcgtttctcaattgaggaacccctagtgatggagttggccactccctctctgc GCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCeCGACGCCCGGGCTTTGCCCGG GCGGCCTCAGTGAGCGAGCGAGCGCGCAG (SEQ ID No. 16) pZac2.1 -ABCA4_3'TS_SV40

Full length sequence of pZac2.1-ABCA4_3'TS_SV40

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGG TCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGG GIT CC TGATAG GCACCIAT TGGTCT TACTGACAT GCACIΤ TGCC ΤIT C TCICCACAGG T CCA TCCTGACGGGTCTGTTGCCACCAACCTCTGGGACTGTGCTCGTTGGGGGAAGGGACATTGAA ACGAGGCTGGATGCAGTCCGGCAGAGeCTTGGCACGTGTCCACAGCACAACATCCTGTTCCA CCACCTCAGGGTGGCTGAGCACATGCTGTTCTATGCCCAGCTGAAAGGAAAGTGCGAGGAGG AGGCeCAGCTGGAGATGGAAGCCAIGTTGGAGGACACAGGCCTCCACCACAAGCGGAATGAA GAGGCTeAGGACCTATCAGGTGGCATGCAGAGAAAGCTGTCGGTTGCCATTGCCTTTGTGGG AQATGCCAAGGTGGTGATTC TGGACGAACC CACC TC TGGGGT GGACCCΤIACTCGAGACGC T CAATCTGGGATCTGCTCCTGAAGTATCGCTCAGGCAGAACCATCATCATGTCCACTCACCAC ATGGACGAGGCCGACCTCCTTGGGGACCGCATTGGCATCATTGCCCAGGGAAGGCTCTACTG CTCAGGCACCCCACTCTTCCTGAAGAACTGCTTIGGCACAGGCTTGIACTTAACCITGGIGC GCAAGATGAAAAACATCCAGAGCCAAAGGAAAGGCAGTGAGGGGACCTGCAGCTGCTCGTCT AAGGGTTTCTCeACCACGTGTCCAGCeCACGTCGATGACCTAACTCCAGAACAAGTCCTGGA TGGGGATGTAAATGAGCTGATGGATGTAGTTCTC.CACCATGTTCCAGAGGCAAAGCTGGTGG AG T GCAT T GGT CAAGAAGΤ TATCTTCC Τ T C TTCCAAATAAGAACΤ T CAAGCACAGAGCATAT gccagccttttcagagagctggaggagacgctggctgaccttggtctcagcagtittggaat itctgacactcccctggaagagattttictgaaggtcacggaggattctgaticaggacctc TGTTTGCGGGTGGCGCTCAGCAGAAAAGAGAAAACGTCAACCCCCGACACCCCTGCTTGGGT

CCCAGAGAGAAGGCTGGACAGACACCCCAGGACTCCAATGTCTGCTCCCCAGGGGCGCCGGe TGCTCACCCAGAGGGCCAGCCTCCCCCAGAGCCAGAGTGCCCAGGCCCGCAGCTCAACACGG GGACACAGC T GG T C CTCCAGCATGTGCAGGCGCTGC TGGT CAAGAGATIC CAACACACCATC CGCAGCCACAAGGACTTCCTGGCGCAGATCGTGCTCCCGGCTACCTTTGTGTTTTTGGCTCT GATGCTTTGTATTGTTATCCCTCCTTTTGGCGAATACCCCGCTTTGACCCTTCACCCCTGGA tatatgggcagcagtacaccttcttcagcatggatgaaccaggcagtgagcagttcacggta cttgcagacGtcctcctgaataagccaggctttggcaaccgctgcctgaaggaagggtggCt T CC GGAG TAC CC C T GTGGCAAC TCAACAC C C T GG AAGAC T CCIT CI GT GT CC CCAAACATCA CCGAGCTGTTCCAGAAGCAGAAATGGACACAGGTCAACCCTTCACCATCCTGCAGGTGCAGC ACCAGGGAGAAGCTCACCATGCTGCCAGAGTGCeCCGAGGGTGCCGGGGGCCTCCCGCCCCC CCAGAGAACACAGCGCAGCACGGAAATTCTACAAGACCTGACGGAC AjGGAACATCTCCGACI TCITGGTAAAAACGIATCCIGCTCTTATAAGAAGCAGCTTAAAGAGCAAATTCIGGGTCAAT GAACAGAGGTATGGAGGAATΤTCCATTGGAGGAAAGCTCCCAGTCGTCCCCATCACGGGGGA agcacTtgttgGgtttttaagcgaccTtggccggatcatgaatgtgagcgggggcgctatga CTAGAGAGGCCTCTAAAGAAATACCTGATTTCCTTAAACATCTAGAAACTGAAGACAACATI aaggtgtggtttaataacaaaggctggcatgccctggtcagctttctcaatgtggcccacaa CGCCATCTTACGGGCCAGCCTGCCTAAGGACAGAAGCCCCGAGGAGTATGGAATCACCGTCA TTAGCCAACCCCTGAACCTGACCAAGGAGCAGCTCTCAGAGATTACAGTGCTGACCACTTCA gtggatgctgtggttgccatctgcgtgattttctccatgtccttcgtcccagccagctttgt gctttatttgatccaggagcgggtgaacaaatccaagcacctccagtttatcagtggagtga gccccaccacciactgggtaaccaacttcctctgggacatcatgaattattccgtgagtgct GGGCTGG TGGT GGGCAT CTT C AT C GGGT Τ T CAGAAGAAAGCC TACAC TTCICCAGAAAAC CT tcctgcccttgtggcactgctcctgctgtatggatgggcggtcattcccatgatgtacccag catccttcctgtttgatgtccccagcacagcctatgtggctttatcttgtgctaatctgttc atcggcatcaacagcagtgctattaccttcatcttggaattatttgagaataaccggacgct GCTCAGGTTCAACGCCGTGCTGAGGAAGCTGCTCATTGTCTTCCCCCACTTCTGCCTGGGCe GGG GCCT C AT T GAC CTIGCAC ΤGAGCCAGGC T GT GACAGATGTC TATGCC C GGTTTGGTGAG GAGCACTCTGCAAATCCGTTCCACTGGGACCTGATTGGGAAGAACCTGTTTGCCATGGTGGT GGAAGGG G T GGT G TACITCCTCCT GAC C C T G C T GGT CCAGCGCCACTTCΤ T CCICICCCAAT ggattgccgagccCagtaagGagcccattgttGatgaagatGatgatGtgggtgaagaaaga CAAAGAATTATTACTGGIGGAAATAAAACTGACATC TTAAGGCTACATGAACTAAOCAAGAT IIATCCAGGCACCTCCAGCCCAGCAGTGGACAGGCTGTGTGTCGGAGTTCGCCCTGGAGAGT GCITTGGCCTCCTGGGAGTGAATGGTGCCGGCAAAACAACCACATTCAAGATGCTCACTGGG GACACCACAGTGACCTCAGGGGATGCCACCGTAGCAGGCAAGAGTATTTTAACCAATATTTe TGAAGTCCATCAAAAIATGGGCTACTGTCCTCAGTTTGATGCAATCGATGAGCTGCTCACAG GACGAGAACATCTTTACCTTTATGCCCGGCΤTCGAGGTGTAGCAGCAGAAGAAATCGAAAAG GTTGCAAACTGGAGTATTAAGAGCCTGGGCCTGACTGTCTACGCCGACTGCCTGGCTGGCAC GTACAGTGGGGGCAACAAGCGGAAACTCTCCACAGCCATCGCACTCATIGGCTGGCCACCGC TGGTGCTGCTGGATGAGCCCACCACAGGGAIGGACCCCCAGGCACGCCGCATGCTGIGGAAC GTCATC GIGAGCATC AT CAGAGAAGGGAGCGCIGTGGT CC TCACAT GC CACAGCATGGAAGA AIGTGAGGCACTGIGTACCCGGCTGGCCATCATGGIAAAGGGCGCCTTTCGAIGTATGGGCA CCATTCAGCATCTCAAGTCCAAATTTGGAGATGGCTATATCGTCACAATGAAGATCAAATCC CC GAAGGAC GAC C T GC Τ T C C T GACCIGAAC CCTGTGGAGCAGΤ T CTTC CAGGGGAACI TCCC AGGCAGTGTGCAGAGGGAGAGGCACTACAACATGCTCCAGTTCCAGGTCTCCTCCTCCTCCC iggcGaggaCcitccagctcctcctctcccacaaggacagcctgctcatcgaggagiacTCa GIGACACAGAC CACAC T GGAC CAGGT GΤ Τ TG TAAATΤITGCTAAACAGCAGAC T GAAAGTCA igacciccctctgcaccctcgagctggtggagccagtcgacaagcccaggacigagcggccg C ΤICGAGGAGACATGATAAGATACATTGATGAGT TIGGACAAACCACAAC TAGAAT GCAGT G AAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCT gcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggagatg TGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGATCTTCC tagagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaaccccta GTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAA GGTCGGCCGACGCCCGGGCTTTGCCCGGSCGGCCICAGTGAGCGAGCGAGCGCGCAG (SEQ ID No. 17)

MYO7A

pAAV2.1 -CBA-MYO7A_5'AK 5' ITR2 AGGAACCCCTAGTGATGGAGΤTGGCCACICCCTCTCTGGGCGCTCGCTCGCTCACTGAGGCC GGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGC GCGCAG (SEQ ID No. 10) LEFT ITR5 CTCTCCCCCCTGTCGCGTTCGeTCGCTCGGTGGCTCGTTTGGGGGGGTGGCAGCTCAAAGAG CTGCCAGACGACGGCCCTCTGGCCGTCGCCCCCCCAAACGAGCCAGCGAGCGAGCGAACGCG' ACAGGGGGGAGAGTGCCACACTCTCAAGCAAGGGGGTTTTGTAAGCAGTGA (SP.Q ID No. 18) CMV enhancer gctaGcgtgccacctggtcgacattgattattgactagttattaatagtaatcaattacggg GTGATTAGTTCATAGCCCATATATGGAGTTCCGGGTTACATAACTTACGGTAAATGGCCCGC CT G GCT GAC C GCC CAAC GAC CCCC GC CCAT T GAC GTCAATAAT GACGTAT GT TCCCATAGTA ACGCCAATAGGGACTTTCCATTGAGGTCAATGGGTGGACTATTTACGGTAAACTGeCCACTT GGCAGTACATGAAGTGTATCATATGCCAAGTACGCeCeCTATTGACGTCAATGACGGTAAAT GGCCCGC CTGGCATTATGCGGAGTACATGACCΤTATGGGACT Τ TCC TACT TGGCAGTACAT C TACGTATTAGTCATCGCTATTACCATGG (SEQ ID No. 19) CBA promoter TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATT ttgtatttatttattttttaattattttgtgcagcgatgggggcGgggGgggggggggcgcg CGCCAGGCGGGGCGGGGCGGGGCGAGGGGGGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCA GCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGeC CTATAAAAAGCGAAGCGCGCGGCGGGCGG (SEQ ID No. 20) SV40 intron

GTAAGTATCAAGGTTAGAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACA GAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCGACTTTGCGTTTe TCTCCACAG (SEQ ID No, 21)

5'hMYO7A CDS

ATGGTGAT T C T TCAGCAGGGGGACCATGTGTGGATGGACCTGAGATT GGC GCAG GAGT T CGA CGTGCCCATCGGGGCGGTGGTGAAGCTCTGCGACTCTGGGCAGGTCCAGGTGGTGGATGATG AAGACAATGAACACTGGATCTCTeCGeAGAACGCAACGCACATeAAGeCTATGCACeCCACG TCGGTCCACGGCGTGGAGGACAIGATCCGCCTGGGGGACCTCAACGAGGCGGGCATCTTGCG CAACCTGCTTATCCGCTACCGGGACCACCTCATCTACACGTATACGGGCTCCATCCTGGTGG CTGTGAACCCCTACCAGCTGCTCTCCATCTACTCGCCAGAGCACATCCGCCAGTATACCAAC AAGAAGATTGGGGAGATGCCCCCCCACATCTTTGCCATTGCTGACAACTGCTACTTCAACAT gaaacgcaacagccgagaccagtgctgcatcatcagtggggaatctggggccgggaagacgg AGAGCACAAAGCTGATCCTGCAGTTCCTGGCAGCCATCAGTGGGCAGCACTCGTGGATTGAG CAGCAGGTCTTGGAGGCCACCCCCATTCTGGAAGCATTTGGGAATGCCAAGACCATCCGCAA TGACAACTCAAGCCGTITCGGAAAGTACATCGACATCCACTTCAACAAGCGGGGCGCCATCG agggcgcgaagattgagcagtacctgctggaaaagtcacgtgtctgtcgccaggccctggat gaaaggaactaccacgtgttctactgcatgctggagggcatgagtga.ggatcagaagaagaa GCT GGGC T TGGGCCAGGCC TC T GACTACAACTAC TTGGCCATGGGTAACTGCATAACC TGTG AGGGCCGGGTGGACAGCCAGGAGTACGCCAACATCCGCTCCGCCATGAAGGTGCTCATGTTC ACTGACACCGAGAACTGGGAGATCTCGAAGCTCCTGGCTGCCATCCTGCACCTGGGCAACCT GGAGTATGAGGCACGGACATT TGAAAACC TGGATGCCTGTGAGGTT CT CT TC TC CCCAT CGC

TGGCCACAGCTGCATCCCTGCTTGAGGTGAACCCCCCAGACCTGATGAGCTGCCTGACTAGC CGCACCCTCATCACCCGCGGGGAGACGGTGTCCACCCCACTGAGCAGGGAACAGGCACTGGA CGTGCGCGACGCCTTCGTAAAGGGGATCTACGGGCGGCTGTTCGTGTGGATTGTGGACAAGA T CAACGCAGCAAT T TACAAGC C TCCC TCCCAGGATGTGAAGAAGTCTCGCAGGTC CAT CGGC CI GCTGGACATCIITGGGTΤTGAGAACIΤIGCIGIGAACAGCIΤ TGAGCAGCICIGCATCAA CTTCGCCAATGAGCACCTGCAGCAGTTCTTTGTGCGGCACGTGTTCAAGCTGGAGCAGGAGG AATATGAC CIGGAGAGCAIIGACT GGCIGCACAT CGAGIICAC T GACAAC C AGGATGCC CIG gacatgattgccaacaagcccatgaacatcatctccctcatcgatgaggagagcaagttccc CAAGGGCACAGACACCACCATGTIACACAAGCTGAACTCCCAGCACAAGC!CAACGCCAACI ACATCCCeCCCAAGAACAACCATGAGACCCAGTTlGGCATCAACCATITTGCAGGCATCGlC TAG TAT GAGAC C CAAGGCT TCC TGGAGAAGAACC GAGACAC CC T GCAT GGGGACAT TAT CCA GeTGGTCCACTCCTCCAGGAACAAGTTCATCAAGCAGATCTTCCAGGCCGATGTeGCCATGG GCGCCGAGAeCAGGAAGCGCTCGCCCACACTTAGCAGeCAGTTCAAGCGGTCACTGGAGCTG CTGATGCGCACGCTGGGTGCCTGCCAGCCCTTCTTTGTGCGATGCATCAAGCCCAATGAGIT CAAGAAGCCCATGCTGTTCGAeCGGCACCTGTGCGTGCGCCAGCTGCGGTACTCAGGAATGA TGGAGACCATCCGAATCCGCGGAGCTGGCTACCCCATCCGCTACAGCITCGTAGAGTTTGTG gagcggtaccgigtgctgctgccaggigtgaagccggcctacaagcagggcgacciccgcgg gacitgccagcgcatggcigaggctgtgctgggcacccacgatgactggcagataggcaaaa ccaagatctttctgaaggaccaccatgacatgctgctggaagtggagcgggacaaagccatc accgacagagtcatcctccttcagaaagtcaiccggggattcaaagacaggtciaactttct GAAGCIGAAGAACGCIGCCACACTGATCCAGAGGCACTGGCGGGGTCACAACTGIAGGAAGA ACTACGGGCTGATGCGTCTGGGCTTCCTGCGGCTGCAGGCCCIGCACCGCTCCCGGAAGCTG CACCAGCAGTACCGGCTGGCCCGCCAGCGCATCATCCAGTTCCAGGCCCGCTGCCGCGCCTA TCIGGTGCGCAAGGGCTTCCGCCACCGCCTCTGGGCTGTGClCACCGTGCAGGCCTATGCCC ggggcatgatcgcccgcaggctgcaccaacgccicagggctgagtatctgtggcgcctcgag gctgagaaaatgcggctggcggaggaagagaagciicggaaggagaigagcgccaagaaggc caaggaggaggccgagcgcaagcatcaggagcgcctggcccagciggctcgtgaggacgctg agcgggagctgaaggagaaggaggcggctcggcggaagaaggagctcctggagcagatggaa AGGGGCCGCCAIGAGCGTGTCAATCACTCAGACATGGTGGACAAGATGTTTGGCTTCCTGGG gacttcaggtggcctgccaggccaggagggccaggcacciagiggctttgaggacctggagt GAGGGCGGAGGGAGATGGTGGAGGAGGACCTGGATGCAGCCCTGCCCCTGCCIGACGAGGAI gaggaggacctcictgagtataaatttgccaagitcgcggccacciacticcaggggacaac TACGCACTCCTACACCCGGCGGGCACTCAAACAGCCACTGCICTACCATGACGACGAGGGTG ACCAGCTG (SEQ ID No.. 22)

Splice donor signal GTAAGTAICAAGGTTACAAGACAGGITTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACA GAGAAGACTCTTGCGTTTCT (SEQ ID No. 1)

AK GGGATT TTGG CGATTT CGGCC TAT TGGTTAAAAAAT GAGC TGAT TTAACAAAAAT Τ TAACGC GAATTTTAACAAAAT (SEQ ID No. 3) 3’ITR2 ctgcgcgctcgcicgctgactgaggCcgccCggGcaaagcccgggcgtcgggcgacctttgg TCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGG GTTCCT (SEQ ID No. 4)

Full-sequence of pAAV2.1-CBA-MYO7A_5’AK

CTGCGCGClCGGTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGICGGGCGACCTITGG TCGCCCGGCCTCAGTCAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGG G Τ T C C T T GT AG ΤI AAT G AT T AACCCGCC AT GCT ACT TACC T ACGT AGCC ATGC TC TAGGAAG AT C C T AAT GGGG AAT T GGCCCT T AAG GT AGGG T GCC AC GT GGTGG AC AT TC-AT TAT T GACTA gttattaatagiaatcaattacggggicattagttcatagcccataiatGgAgticcgcgtt acataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtc aataatgaggtatgttcccatagtaaggccaaiagggactttccattgacgtcaatgggtgg ACTATTIACGGTAAACTGCCCACTIGGCAGTACATCAAGTGTAICATATGCCAAGIACGCCC CCTATTGAGGTCAATGACGGTAAATGGCCCGGGTGGGATTATGCCCAGTACATGAGCTTATG GGAGT TTGG TACTTGGCAGTACATGIAGGTAT TAGTGATCGCTAT TACGATGGGTCGAGGTG AGC C CCACGTTC TGC Τ TC ACT C T C CC C AT C T CC CC CC CCTCCCCACCCCCAATTTTGTAT TT ATTTATTTTTTAATTATTTTGTGCAGGGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGC GGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCA GAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAA

AGCGAAGCGCGCGGCGGGCGGCTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCA

AGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGICGAGACAGAGAAGACTC GTGCGCTTCTGATAGGCACCTATTGGTCTTAGTGACATCCACTTTGCCTTTCTCTCCACAGG'

TGTCCAGGCGGGCGCCATGGTGATTCTTCAGCAGGGGGACCATGTGTGGATGGACCTGAGAT TGGGGCAGGAGTTCGACGTGCCCATCGGGGCGGTGGTGAAGCTCTGCGACTCTGGGCAGGTC:

CAGGTGGTGGATGATGAAGACAATGAACACTGGATCTCCCCGCAGAACGCAACGCACATCAA GCCTATGCACCCCACGTCGGTCCACGGCGTGGAGGACATGATCCGCCTGGGGGACCTCAACG AGGCGGGCATCTTGCGCAACCTGCTTATCCGCTACCGGGACCACCTCAICTACACGTATACG GGCTCCATCCTGGTGGCTGT&amp;AACCCCTACCA&amp;rTGCTTTCCATGTACTCGCCAGAGCACAT gcgccagtataccaagaagaagattggggagatgggggggcaCatctttgggattggtgaca actgctacttcaacatgaaacgcaacagccgagaccagcgctgcaTcatgagtggggaatct ggggccgggaagacggagagcacaaagctgatcctgcagttcctggcagccatcagtgggca ccactcctccattcaccagcacgtcttccaccccacccccatictcgaaccatttggcaatc CCAAGACCAT CCGCAATGAGAACT CAAGC CGΤ Τ TCGGAAAGIAGATCGACATCCACΤ T CAAC AAGCGGGGCGCCATCGAGGGCGCGAAGATTGAGCAGTACCTGCTGGAAAAGTCACGTGTCTG T C GCCAGGCCCTGGATGAAAGGAACIACCACGT GT TCIACTGCATGCIGGAGGGCATGAGTG AGGATCAGAAGAAGAAGCTGGGCTTSGGCCAGGCCCCTGACTACAACTACTTGGCCATGGGT AACTGCATAACGTGIGAGGGCCGGGTGGACAGCCAGGAGTACGCCAACATCCGCTGCGCCAT GAAGGTGCTCATGTTCACTGAeACCGAGAACTGGGAGATCTCGAAGCTCCTGGCTGCCATCC tgcacctgggcaagctgcagtatgaggcacgcacatttgaaaacctggatgcctgtgaggtt CTCTTCTCCCCATGGCTGGCCACAGCTGCATCCCTGCTTGAGGTGAACCCCCCAGACCTGAT GAGCTGCCTGACTAGCCGCACCCTCATCACCCGCGGGGAGACGGTGTCCACCCCACTGAGCA GGGAACAGGCACTGGACGIGCGCGACGCGTTCGTAAAGGGGATCTACGGGCGGCTGTTCGTG: TGGATTGTGGACAAGATCAACGCAGCAATTTACAAGCCTCCCTCCCAGGATGTGAAGAACTC TCGCAGGTCCATCGGCCTCCTGGACATCTTTGGGTTTGAGAACTTTGCTGTGAACAGCTTTG: AGCAGCTCTGCATCAACTTCGCCAATGAGCACCTGCAGCAGTTCTTTGTGCGGCACGTGTTC AAGGTGGAGCAGGAGGAATATGACCTGGAGAGCATTGACTGGCTGCACATCGAGTTCACTGA CAACCAGGATGCCCTGGACATGATTGCCAACAAGCCCATGAACATCATCTCCCTCATCGATG AGGAGAGCAA&amp;T tc C CCAAGGGCACAGACAC CACCATG t tacacaagcτgaactcccagcac AAGCTCAACGCCAACTACATCCCCCCCAAGAACAACCATGAGACCCAGTTTGGCATCAACCA TTTTGCAGGCATCGTCT AC TAT GAGACCCAAGGCΤTCCTGGAGAAGAAGGGAGACACCCTG C atggggacattatccagctggtccactcctccaggaacaagttcatcaagcagatcttccag: GCCGATGTCGCCATGGGCGCCGAGACCAGGAAGCGCTCGCCCACACTTAGCAGCCAGTTCAA GCGGTCACTGGAGCTGCTGATGCGCACGCTGGGTGCCTGCCAGCCCTTCTTTGTGCGATGCA TCAAGCCC.AAIGAGTTCAAGAAGCCCATGCTGTTC.GACCGGCACCTGTGC.GTGCGCCAGCTG CGGTACTCAGGAATGATGGAGACCATCCGAATCCGCCGAGCTGGCTACCCCATCCGCTACAG OTTCGTAGAGTTTGTGGAGCGGTACGGTGTGCTGCTGOCAGGTGTGAAGCCGGCGTACAAGO AGGGCGACCTCCGCGGGACTTGCCAGCGCATGGCTGAGGCTGTGCTGGGCACCCACGATGAC tggcagatagggaaaacCaagatctttctgaaggaccaccatgacatGctgctggaagtgga gcgggacaaagccatcaccgacagagtcatcctgcttcagaaagtcatccggggattgaaag acaggtctaactttctgaagctgaagaacgctgccacactgatccagaggcactggcggggt CACAACTGTAGGAAGAACTACGGGCTGATGCGTCTGGGCTTCCTGCGGCTGCAGGCCCTGeA CCGCTCCCGGAAGCTGCACCAGCAGTACCGCCTGGCCCGCCAGCGCATCATCCAGTTCCAGG CCCGCTGCCGCGCCTATCTGGTGCGCAAGGCCTTCCGCCACCGCCTCTGGGCTGTGCTCACC gtgcagggctatgcccggggcatgatcgcccgcaggctgcacgaacgcctcagggctgagta tcigtggcgcctcgaggctgagaaaatgcggctggcggagGaagagaagcttcggaaggaga tgagcgccaagaagggcaaggaggagggcgagcgcaagcatcaggagcgcctggcccagctg GCTGGTGAGGAGGCTGAGCGGGAGGTGAAGGAGAAGGAGGCCGCTCGGCGGAAGAAGGAGCT CCTGGAGGAGATGGAAAGGGCCCGCCATGAGCCTGTCAATCACICAGACATGGTGGACAAGA TG T Τ TGGG T T C C T GGGG AC TTCAGGTGGCC TGCCAGGCCAGGAGGGCCAGGCACCTAGTGGC TTTGAGGACCTGGAGCGAGGGCGGAGGGAGATGGTGGAGGAGGACeTGGATGCAGCCCTGCC CCTGCCTGACGAGGATGAGGAGGACCTCTCTGAGTATAAATTTGCCAAGTTCGCGGCCACCT acttccaggggacaactacgcactcctacacccgggggccactcaaacagccactgctctac catgacgacgagggtgaccagctggtaagtatcaaggttacaagacaggtttaaggagacca atagaaactgggcttgtcgagacagagaagactcttgcgtttctgggattttgccgatttcg GCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATΤTTAACAAAATATT AACGTTTATAATTTCAGGTGGCATCTTTCCAATTGAAGGGCGAATTCCGATCTTCCTAGAGC ATGGOTACGTAGATAAGTAGCATGGCGGGTTAAICATTAACTACAAGGAACCCCTAGIGATG GAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGC CCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG (SEQ ID No. 23)

pAAV2.1 -MYO7A_3'AK_BGH 5' ITR2 AGGAACGC C T AG TGATGGAGTTGGC CACTCCCTCTC TGCGCGC TCGCTC GCTCACTGAGGCC GGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGC GCGCAG (SEQ ID No. 10)

AK gggattttgcggatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgc GAATTTTAACAAAAT (SEQ ID No. 3)

Splice acceptor signal GATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG (SEQ ID No. 1)

3'hMYO7A CDS

gcagccctggcggtctggatcaccatcctccgcttcatgggggacctccctgagcccaagta eCACACAGCCATGAGTGATGGCAGTGAGAAGATCCCTGTGATGACCAAGATTTATGAGAeCC T GGGCAAGAAGACGTACAAGAGGGAGC T GCAGGCCC TGCAGGGCGAGGGCGAGGCCCAGCTC CCCGAGGGCCAGAAGAAGAGCAGTGTGAGGCACAAGCTGGTGCATTTGACTCTGAAAAAGAA gtccaagctcacagaggaggtgaccaagaggctgcatgacggggagtccacagtgcagggga ACAGCATGCTGGAGGACCGGCCCACCTCCAACCTGGAGAAGCTGCACTTCATCATCGGCAAT GGCATCCTGCGGCCAGCACTCCGGGACGAGATCTACTGCCAGATCAGCAAGCAGCTGACCCA CAACCCCTCCAAGAGCAGCTATGCCCGGGGCTGGATTCTCGTGTCTCTCTGCGTGGGCTGTT TCGCCCCCTCCGAGAAGTTTGTCAAGTACCTGCGGAACTTCATCCACGGGGGCCCGCCCGGC

tacgccccgtacGgTgaggagcgcctgagAaGgacctttgtcaatgggacacggacacagcc gcccagctggctggagctgcaggccaccaagtccaagaagccaatcatgttgcccgtgacat tcatggatgggaccaccaagaccctgctgacggactcggcaaccacggccaaggagctctgc AACGCGCTGGCCGACAAGATCTCTCTCAAGGACCGGTTCGGGTTCTCCCTCTACATTGCCCT GTTTGACAAGGTGGCCTCCCTGGGCAGCGGCAGGGACCACGTCATGGACGCCATCTCCCAGG gcgagcagtacgccaaggagcagggcgcccaggagcgcaacgccccctggaggctcttcttc CGCAAAGAGGTCTTCACGCCCTGGCACAGCCCCTCCGAGGACAACGTGGCCACCAACCTCAT ctaccagcaggtggtgcgaggagtcaagtttggggagtacaggtgtgagaaggaggacgacc TGGCTGAGCTGGCCTCCCAGCAGTACTTTGTAGACTATGGCTCTGAGATGATCCTGGAGCGC CTCCTGAACCTCGTGCCCACCTACATCCCCGACCGCGAGATCACGCCCCTGAAGACGCTGGA GAAGTGGGCCCAGCTGGCGATCGCCGCCCACAAGAAGGGGATTTATGCCCAGAGGAGAACTG ATGCCCAGAAGGTCAAAGAGGATGTGGTCAGTTATGCCCGCTTCAAGTGGCCCTTGCTCTTC T CCAGGTT T TATG AAGCC TAGAAATICT CAGGCCCCAGT C TCCCCAAGAACGACGTCATCGT GGCCGTCAACTGGAeGGGTGTGTACTTTGTGGATGAGCAGGAGCAGGTACTTGTGGAGGTGT CCTTCCCAGAGATCATGGCCGTGTCCAGCAGCAGGGAGTGCCGTGTCTGGCTCTCACTGGGC TGC TCT GAT CT T GGC TGT GCT GC GC C TCACT CAGGC TGGGCAGGACTGACCCCGGCGGGGCC CT G Τ T C TCCGTGT T GG TC CTGCAGGGGAGCGAAAACGACGGC C C C C AGC.T T C AC GC T GGC C A CCATCAAGGGGGACGAATACACCTTCACCTCCAGTAATGCTGAGGACATCCGTGACCTGGTG GTCACCTTCCTAGAGGGGCTCCGGAAGAGATCTAAGTATGTTGTGGCCCTGCAGGATAACCC CAACCCCGCAGGCGAGGAGTCAGGCTTCCTCAGCTTTGCGAAGGGAGACCTCATCATCCTGG ACCATGACACGGGCGAGCAGGTCATGAACTCGGGCTGGGCCAACGGCATCAATGAGAGGACC AAGCAGCGTGGGGACTTCCCCACCGACTGTGTGTACGTCATGCeCACTGTCACCATGCCACC TCGTGAGATTGTGGCCCTGGTCACCATGACTCCCGATCAGAGGCAGGACGTTGTCCGGCTCT TGCAGCTGCGAACGGCGGAGCCCGAGGTGCGTGCCAAGCCCTACACGCTGGAGGAGTTTTCC TATGACTACTTCAGGCCCCCACCCAAGCACACGCTGAGCCGTGTCATGGTGTCCAAGGCCCG AGGCAAGGACCGGCTGTGGAGCCACACGCGGGAACCGCTCAAGCAGGCGCTGCTCAAGAAGC TCCTGGGCAGTGAGGAGCTCTCGCAGGAGGCCTGCCTGGCCTTCATTGCTGTGCTCAAGTAC ATGGGCGACTACCCGTCCAAGAGGACACGCTCCGTCAATGAGCTCACCGACCAGATCTTTGA GGGTCCCeTGAAAGCCGAGCCCCTGAAGGACGAGGCATATGTGCAGATCCTGAAGCAGCTGA CCGACAAeCACATCAGGTACAGCGAGGAGCGGGGTTGGGAGCTGCTCTGGCTGTGCACGGGC CTTTTCCCACCCAGCAACATCCTCCTGCCCCACGTGCAGCGeTTCeTGeAGTCCCGAAAGCA CTGCGCACTCGCCATCGACTGCCTGe^AACGGCTeCAGAAAGCCCTGAGAAACGGGTCCCGGA AGTACCCTCCGCACCTGGTGGAGGTGGAGGCCATCCAGCACAAGACCACCCAGATTTTCCAC AAGC TCTAGΤ T CCCT CAT C ACAC T C AC GAC C CC Τ T CGAAGT GG AGT CGAG CAGCAAGCCC AA GGACTTCTGCCAGAACATCGCCACCAGGCTGCTCCTCAAGTCCTCAGAGGGATTCAGCCTCT TTGTCAAAATTGCAGACAAGGTCATCAGCGTTCCTGAGAATGACTTCTTCTTTGAeTTTGTT cgacacttgacagactggataaagaaagctcggcccatcaaggacggaattgtgccctcact cacctacgaggtgttcttcatgaagaagctgtggaccaccacggtgccagggaaggaiccca tggccgattccatcttccactattaccaggagttgcccaagtatciccgaggciaccacaag tgcacgcgggaggaggtgctgcagctgggggcgctgatctacagggtcaagttcgaggagga caagtcctacttccccagcatccccaagctgctgcgggagctggtgccccaggaccttatcc ggcaggtctcacctgatgactggaagcggtccatcgtcgcctacttgaacaagcacgcaggg aagtccaaggaggaggccaagctggccttcctgaagctcatcttcaagtggcccacctttgg ctcagccttcttcgaggtgaagcaaactacggagccaaacttccctgagatcctcctaattg ccatcaacaagtatggggtcagcctcatcgatccgaaaacgaaggatatgctgaccactgat C C C Τ T C AC CAAGATCTCCAACTGGAGCAGC GGCAACAGCTAC Τ T C CACATCACCATTGGGAA CTTGGTGCGCGGGAGCAAACTGCTCTGCGAGACGTCACTGGGCTACAAGATGGATGACCTCC tgacttcctacattaggcagatgctgacagccatgaGcaaacagcggggctcgaggagcggc AAGTGA (SEQ ID No. 24)

BGH poly A gcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttcctt gaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcatt gtctgagtaggtgtcattctattctggggggtgggGtggggcaggacagcAagggggaggat TGGGAAGACAATAGCAGGCATGCTGGGGA (SEQ ID No. 25) 3'ITR2 CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGG TCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGG GTTCCT (SEQ ID No. 4) RIGHT ITR5 tcactgcttaCaaaacccccttgcttgagagtgtggcactCtcccccctgtcgcgttcgctc gctcgctggctcgtttgggggggcgacggccagagggccgtcgtctggcagctctttgagct GCCACCCCCCCAAACGAGCCAGCGAGCGAGCGAACGCGACAGGGGGGAGAG (SEQ ID No. 14)

Full-sequence of pAAV2.1-MYO7A_3'AK_BGH

ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttgg TCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCGAACTCCATCACTAGGG gticctigtagttaatgaiiaacccggcaigctacttatctacgiagccaigctciaggaag atgggaattcgccctttgatcagggattttgccgatttcggcctattggttaaaaaatgagc TG ATTTAACAAAAATTTAACGCGAATT TT AACAAAATATTAAGGTTTATAAT TICAGGT GGC ATCTTTCGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTGTCCACAGGCAG ccciggcggtctggatcaccatcctccgcttcatgggggacctccctgaggccaagtaccac ACAGCCATGAGTGATGGCAGTGAGAAGATCCCTGIGATGAGCAAGATTTATGAGACCeTGGG CAAGAAGACGTACAAGAGGGAGCTGCAGGCCCTGCAGGGCGAGGGCGAGGCCCAGCTeCCCG AGGGCCAGAAGAAGAGCAGTGTGAGGCACAAGCTGGTGCATTTGACTCTGAAAAAGAAGTCC AAGCTCACAGAGGAGGTGACCAAGAGGCTGCATGACGGGGAGTCCACAGTGCAGGGCAACAG CATGCTGGAGGACCGGCCCACCTCCAACCTGGAGAAGCTGCACTTCATCATCGGCAATGGeA TCCTGCGGCCAGCACTCCGGGAGGAGATCTACTGCCAGATCAGCAAGCAGCTGACCCACAAC CCCICCAAGAG CAGCTATGCCCGGGGC T GG AT T C TCGTGTCTC TC TGCGT GGGCTGT TTCGC CCCCTCCGAGAAGTTTGTCAAGTACCTGCGGAACTTCATCCACGGGGGCCCGCCCGGCTACG CCCCGTACTGTGAGGAGCGCCTGAGAAGGACCTTTGTCAATGGGACACGGACACAGCCGeCC AGCTGGCTGGAGCTGCAGGCCACCAAGTCCAAGAAGCCAATCATGTTGCCCGTGACATTCAT GGATGGGACCACCAAGACCCTGCIGACGGACTCGGCAACCACGGCCAAGGAGCTCTGCAACG CGCTGGCCGACAAGATCTCTCTCAAGGACCGGTTCGGGTTCTCCCTCTACATTGCCCTGTTT GACAAGGTGTCCTCCCTGGGCAGCGGCAGTGACCACGTCATGGACGCCATCTCCCAGTGCGA GCAGTACGCCAAGGAGCAGGGCGCCCAGGAGCGCAACGCCCCCTGGAGGCTCTTCTTCCGCA

AAGAGGT CT T CACGCCCTGGCACAGCCCCTCCGAGGACAACGTGGC CACC AACCICATC TAC CAGCAGGT GGT GCGAGGAGTCAAGΤ T TGGGGAGTACAGGT G T GAGAAGGAGGACGAC C T GGC tgagctggcctcccagcagtactttgtagactatggctctgagatgatcctggagcgcctcc TGAACCTCGTGCCCACCTACAT CCC CGACCGGGAGATCACGC C C CT GAAGACGOT GGAGAAG TGGGCCCAGCTGGCCATCGCCGCCCAeAAGAAGGGGATTTATGCCCAGAGGAGAACTGATGe CCAGAAGGTCAAAGAGGATGTGGTCAGTTATGCGCGCTTCAAGTGGCCCTTGCTCTTCTCCA GGTTTTATGAAGCCTACAAATTCTCAGGCCCCAGTCTCCCCAAGAACGACGTCATCGTGGCC GTCAAC T GGAC GGGT GTGTACT TT GT GGAT GAGCAGGAGCAGGTACTTCTGGAGCTGTCCTT CCCAGAGAT CAT GGCCGT GT CCAGCAGCAGGGAGTGCCGT GT C TGGCTCTGACTGGGCΊGCT CTGATCTTGGCTGTGCTGCGCCTCACTCAGGCTGGGCAGGACTGACCCCGGCGGGGCCCTGT TCTC CG TGT T GGTCCT GCAGGGGAGCGAAAACGACGGCCCCCACC TTC AC GC TGGCCACCAT CAAGGGGGACGAATACACCTTCACCTCCAGTAATGCTGACGACATTCGTGACCTGGTGGTCA CCTTCCTAGAGGGGCTCCGGAAGAGATCTAAGTATGTTGTGGCCCTGCAGGATAACCGCAAC CCCGCAGGCGAGGAGTCAGGCTTCCTCAGCTTTGCCAAGGGAGACGTCATCATCCTGGACCA TGACACGGGCGAGCAGGT CAT GAACT CGGGCTGGGCCAACGGCATCAATGAGAGGACCAAGC AGCGTGGGGACTTCCCCACCGACTGTGTGTACGTCATGCTCAGTGTCACCATGCCACCTCGT GAGATTGTGGCCGTGGTCACCATGACTCCGGATCAGAGGCAGGACGTTGTCCGGCTCTTGCA GCTGCGAACGGCGGAGCCCGAGGTGCGTGCCAAGCCCTACACGCTGGAGGAGTTTTCCTATG ACTACTTCAGGCCCCCACCCAAGCACACGCTGAGCCGTGTCATGGTGTCCAAGGCCCGAGGC AAGGACCGGCTGTGGAGCCACACGCGGGAACCGCTCAAGCAGGCGCTGCTCAAGAAGCTCCT gggcagtgaggagctctcgcaggaggcctgcctggccttcattgctgtgctcaagtacatgg gCGactacccgtccaagaggacacgctccgtcaatgagctcaccgaccagatctttgagggt cgcctgaaaggcgagcccctgaaggacgaggcatatgtgcagatcctgaagcagctgaccga CAAeCACATCAGGTACAGCGAGGAGCGGGGTTGGGAGCTGCTGTGGGTGTGCACGGGCCTTT TCC GACC CAGCAACATCCTCCTGCCCCACGT GCAGCGCT TCC T GCAGT CCC GAAAGC AC TGC CCACTCGCCATCGACTGCCTGCAACGGCTCCAGAAAGCCCTGAGAAACGGGTCCCGGAAGTA CCCTCCGCACCTGGTGGAGGTGGAGGCCATCCAGCACAAGACCACCCAGATTTTCC ACAAGG TCTACTTCCCTGATGACACTGACGAGGCCTTCGAAGTGGAGTCCAGCACCAAGGCCAAGGAe TTCTGCCAGAACATCGCCACCAGGCTGCTCCTCAAGTCCTCAGAGGGATTCAGCCTCTTTGT CAAAATTGCAGACAAGGTCATCAGCGTTCCTGAGAATGACTTGTTCTTTGACTTTGCTCGAe ACTTGACAGACTGGATAAAGAAAGCTCGGCCCATCAAGGACGGAATTGTGCCCTeACTCACC TACCAGGTGTTCTTCATGAAGAAGCTGTGGACCACCACGGTGCCAGGGAAGGATCCGATGGC cgattccatcttccactattaccaggagttgcccaagtatgtccgaggctaccacaagtgca CGC GGGAGGAGGTGC T GCAGC T GGGGGC GC TGAT CTACAGGGTCAAGT TC GAGGAGGACAAG T CC T AC T TCCC CAGCATCCCCAAGC T GCT GCGGGAGCT GGTGCCCCAGGACC T TATCCGGCA GGTCTCACCTGATGACTGGAAGCGGTGCATCGTCGCCTACTTCAACAAGCACGCAGGGAAGT CCAAGGAGGAGGCCAAGCTGGCCTTCCTGAAGCTCATCTTCAAGTGGCCCACCTTTGGCTCA GCCTTCTTCGAGGTGAAGCAAACTACGGAGCCAAACTTCCCTGAGATCCTCCTAATTGCCAT CAACAAGTATGGGGTCAGCCTCATCGATCCCAAAACGAAGGATATCCTCACCACTCATCCCT T CACCAAGAT CTCCAACT GGAGCAGCGGCAACACCTACΤTCCACATCACCATT GGGAACT T G G T G C GC G GGAGCAAACTGCTC T GCGAGACGT CAC TGGGCTACAAGAT GCAT GAC C TCCTGAC TTCCTACATTAGCCAGATGCCCACAGCCATGAGCAAACAGCGGGGCTCCAGGAGCGGCAAGT GACCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGAGATCTGCCTCGACTGTG CCT T C TAGTT GCGAGCGATCTGTTGT T TGCCCCTCCCCCGTGCCTT CC TTGACCCTGGAAGG TGCCAGTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGT GTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAAT AGCAGGCATGCT GGGGACTCGAGT TAAGGGCGCAAT TC CCGATTAGGATC T TCC TAGAGCAT GGCTACGTAGATAAGTAGCATGGCGGGTIAATCATTAACTACAAGGAACCCCTAGTGATGGA GTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCC GACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG (SEQ ID No. 26)

pAAV2.1 -CBA-MYO7A_5’TS

Full-sequence

C TG C G CG C T CGC TCGCTCAC TGAGGCCGCCCGGGCAAAGC CCGGGCGT CGGGCGACC TT T GG tcgcccggcctgaGtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggg GTTCGTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTGIAGGAAG ATCCTAATCGGGAATTCGCCCTTAAGCTAGCGTGCCACCTGGTCGACATTGATTATTGACCA

GTTATTAAIAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT

ACATAACTTACGGTAAATGGCCCGCeTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTC

AATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGG agtatttacggtaaactgcgcagttggcagtacatgaagtgtatcatatgggaagtacgccg cctattgacGtcaatgacggtaaatggcccgcctggcattatgcccagtagatgaccttatg

GGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGeTATTACCAIGGGTCGAGGTG AGC C CCACG TTO TGC TTCACTC TCCCCAT CTC CCCCCCCTC CCCACC C C CAAT TTT GTAT Τ T ATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGC ggggcggggcggggcgaggggcggggcggggcgagggggagaggtgcggcggcagccaatca GAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAA AGCGAAGCGCGCGGCGGGCGGC'TGCAGAAGT'TGG'TCGTGAGGCACTGGGCAGG'i'AAG'TATCA AGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTC TTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGG TGT CCAGGC GGCCGCCAT GGTGAT TC T TCAGCAGGGGGAC CATGTG T GGATGGACC TGAGAT tggggcaggagttcgacgtgcccatcggggcggtggtgaagctctgcgactctgggcaggtc CAGGTGGTGGATGATGAAGACAATGAACACTGGATGTCTCCGCAGAACGCAACGCACATCAA GCCTATGCACCGCACGTCGGTCCACGGCGTGGAGGACAOGATCCGCCTGGGGGACCTCAACGi AGGCGGGCATCTTGCGCAACCTGCTTATCCGCTACCGGGAGCACCTCATCTACACGTATACG· GGCTCCATGCTGGTGGCTGIGAACCCCTACCAGCTGCTCTCCATCTACTCGCCAGAGCAGAT CCGCCAGTATACGAACAAGAAGATTGGGGAGATGCCCCCCCACATCΤΤΪGCCATTGCTGACA ACTGCTACTTCAACATGAAACGCAACAGCCGAGACCAGT'GCTGCATCATCAGTGGGGAATCT GGGGCCGGGAASACGGAGAGCACAAAGCTGATCCTGCAGTTCCTGGCAGCCATCAGTGGGCA GCACTCGTGGATTGAGCAGCAGGTGTTGGAGGCCACGGCCATTCTGGAAGCATTTGGGAATG CCAAGACCATCCGGAATGACAACTCAAGCCGTTTCGGAAAGTACATCGACATCCACTTCAAC AAGCGGGGCGCCATCGAGGGGGCGAAkiATTGAGCAGTACCTGCTGGAAAAGTCAOGIGTCTG TCGCCAGGCCCTGGATGAAAGGAACTACCACGTGTTCTACTGCATGCTGGAGGGCATGAGTG AGGATCAGAAGAAGAAGCTGGGCTTGGGCCAGGCCTCTGACTACAACTACTTGGCCATGGGT AACTGCATAACCTGTGAGGGCCGGGTGGACAGCCAGGAGTACGCGAAeATCCGCTeCGCCAT GAAGGTGCTCATGTTCAeTGAeACCGAGAACTGGGAGATCTCGAAGCTCCTGGCTGCCATCC TGCACCTGGGCAACCTGCAGTATGAGGCACGCACATTTGAAAACCIGGATCCCTGTGAGGIT C TCTTCTCCCrATCGC TG&amp;CC ACA&amp;C'TGCATCCC TGCTmG AGGTGAAC CCC GCG GAC rTGAT GAGCTGCCTGACTAGCCGCACCCTCATCACCCGCGGGGAGACGGTGTCCACCCCACTGAGCA GGGAACAGGCACTGGACGTGCGCGACGCCTTCGTAAAGGGGATCTACGGGCGGCTGTTCGTG Τ66ΑΤΤ6ΤΘ3ΑΟΑΑΟΑΤΟΑΑ060Α60ΑΑΤΤΙΑΟΑΑ600ΤΌΟΟΤΟΟΟΑ06ΑΤ6Τ6ΑΑΘΑΑΟΤΟ TCGCAGGTeCATCGGCCTCCTGGACATCTTTGGGTTTGAGAACTTTGCTGIGAACAGCTTTG AGCAGCTCTGCATCAACΤTGGCCAATGAGCACCTGCAGCAGTTCTΤTGTGCGGCACGTGITC AAGCTGGAGCA3GAGGAATATGACCTGGAGAGCATTGACTGGCTGCACATCGAGTTCACTGA CAACCAGGATGCCCTGGACATGATTGCCAACAAGCCCATGAACATCATCTCCCTCATCGATG AGGAGAGCAAGTTCCCCAAGGGCACAGACACCACCATGTTACACAAGCTGAACTCCCAGCAC AAGCTCAACGCOAACTACATCCCCCCCAAGAACAACCATGAGACCCAGTΤTGGCATCAACCA T TTTGCAGGCATCGTCTACTATGAGACCCAAGGCTTCCTGGAGAAGAACCGAGACACC CTGC ATGGGGACATIATCCAGCTGGTCCACTCC TCCAGGAACAAGΤ T C ATCAAGCAGATCTTCCAG GCCGATGTCGCCATGGGCGCCGAGACCAGGAAGCGCTCGCCCACACTTAGCAGOGAGTTCAA gcggtcactggagctgctgatgcgcacgctgggtgcctgccagcccttctitgtgcgatgca TCAAGCCCAATGAGTTCAAGAAGCCCATGCTGTTCGACCGGCACCTGTGCGTGCGCCAGCTG cggtactcaggaatgatggagaccatccgaatccgcggagctggctagcccatccgctacag cttcgtagagtttgtggagcggtaccgtgtgctgctgccaggtgtgaagccggcctacaagc agggcgacctccgcgggacttgccagcgcatggctgaggctgtgctgggcacccacgatgac TGGCAGATAGGCAAAACCAAGATCTTTCTGAAGGACCACCATGACATGCTCCTGGAAGTGGA GCGGGACAAAGCCATCACCGACAGAGTCATCCTCCTTCAGAAAGTCATCCGGGGATTCAAAG ACAGGTC TAAC TTT C TGAAGC T GAAG AAC GC TGC CACAC TGATG CAGAGGCAC TGGCGGGG T CACAACTGTAGGAAGAACTACGGGCTGATGCGTCTGGGCTTCCTGCGGCTGCAGGeCCTGCA CCGCTCCCGGAAGCTGCACCAGCAGTACCGCCTGGCCCGCCAGCGeATCAICCAGTTCCAGG cccgcTgcCGcgcctatctggtgcgcaaggccttccgccaccgcctctgGgctgTgctcacc GTGCAGGCCTATGCCCGGGGCATGATCGCCCGCAGGCTGCACCAACGCCTCAGGGCTGAGTA tctgtggcgcctcgaggctgagaaaaigcggctggcggaggaagagaagcitcggaaggaga

TGAGCGCCAAGAAGGCCAAGGAGGAGGCCGAGCGCAAGCATCAGGAGCGCCTGGCCCAGCTG gctggtgaggacgctgagcgggagctgaaggagaaggagggcgctcggcggaagaaggagct CCTGGAGCAGATGGAAAGGGCCCGCCATGAGCCTGTCAATCACTCAGACATGGTGGACAAGA TGTTTGGCTTCeTGGGGACTTCAGGTGGCCTGCCAGGCCAGGAGGGCCAGGCACCTAGTGGC TTTGAGGACCTGGAGCGAGGGCGGAGGGAGATGGTGGAGGAGGACCTGGATGCAGCCCTGCC cctgcctgacgaggatgaggaggacctctctgagtataaatttgccaagttcgcggccacct ACTTCCAGGGGACAACTACGCACTCCTACACCCGGCGGCCACTCAAACAGCCACTGCTCTAC catgacgacgagggtgaccagciggtaagtatcaaggttacaagacaggtttaaggagacca AT AGAAAGTGGGC TTGTCGAGACAGAGAAGACTCIT G C G TTICICAAITGAAGGGCGAAT T C CGATCTTCCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAG gaacgcciagtgaiggagttggccactccctctcigcgcgcicgctcgctcactgaggccgg gcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagigagcgagcgagcgc GCAG (SEQ TD No. 27)

pAAV2.1 -/WYO7A_3'TS_BGH

Full-sequence CTGeGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACeTTTGG tcgcccggCctcagtgagcgagcgagcgcGcagagagggagtGgccaactccatcactaggg gttccttgtagttaatgattaacccgccatgctacttatctacgtagccatgctctaggaag ATC GGAATTCGATAGGGACCTATTGGTCTTACTGACAT GCAC Τ TTGCCTTIC TCTCCAC AGG CAGCCCT GGCGGTCTGGATCACCATGC TCCGCTT CATGGGGGAGCICGCTGAGC CCAAGTAC cacacagccatgagtgatggcagigagaagaicccigtgatgaccaagatttatgagaccct gggcaagaagacgtacaagagggagctgcaggccctgcagggcgagggcgaggcccagctcg CCGAGGGCCAGAAGAAGAGCAGTGTGAGGCACAAGCTGGTGCATTTGACTCTGAAAAAGAAG tccaagctcacagaggaggtgaccaagagcctgcatgacggggagtccacagtgcagggcaa cagcatgctggaggaccggcccacctccaacctggagaagctgcacttcatcatcgGcaatg gcatcctgcggccagcactccgggacgagatctactgccagatcagcaagcagctgacccac aacccctccaagagcagctaigcccggggctggattctcgtgtctctctgcgtgggctgttt cgccccctccgagaagtttgtcaagtacctgcggaacttcatccacgggggcccgcccggct acgccccgtactgtgaggagggcctgagaaggacctttgtcaatgggacacggacacagccg CCCAGCTGGCTGGAGCTGCAGGCCACCAAGTCCAAGAAGCCAATCATGTTGCCCGTGACATT CATGGATGGGACCACCAAGACCCTGCIGACGGACTCGGCAACCACGGCCAAGGAGCICTGCA ACGCGCTGGCCGACAAGAICTCTCTCAAGGACCGGTTCGGGTTCTCCCTCTACATTGCCeiG tttgacaaggtgtcctccctgggcagcggcagtgaccacgtcatggacgccaictcccagtg cgagcagiacgccaaggagcagggcgcccaggagcgcaacgccccctggaggctccicttcg GCAAAGAGGTCΤTCACGCCC TGGCACAGCCC C TCC GAGGACAAC GT GGCCACCAAC C T CAT C TACCAGCAGGTGGTGCGAGGAGTeAAGTTTGGGGAGTACAGGTGTGAGAAGGAGGACGACCT GGC TGAGCTGGCCTCCCAGCAGTACTITGTAGAC TATGGC T C T GAGAT GAT CC TGGAGCGCC tcctgaacctcgtgcccacciacatccccgaccgcgagatcacgcccctgaagacgctggag AAGIGGGGGCAGCTGGCCATCGCCGCGCACAAGAAGGGGATTTATGGCCAGAGGAGAACTGA iggccagaaggicaaagaggatgtggtcagttatgccggcttcaagtggccciiggtcttct CCAGGTTTTATGAAGCCIACAAATTCTCAGGCCeCAGTCTCCeCAAGAACGACGTCATCGIG GCCGTCAACIGGACGGGTGT GTACT TTGTGGAT GAGCAGGAGCAGGTAGTIC TGGAGCTGIC CTTCCCAGAGATCATGGCCGTGTCCAGCAGCAGGGAGTGCCGTGTCTGGCTCTCACTGGGCT GCTCTGATCTTGGCTGTGCTGCGCCTCACTCAGGCTGGGCAGGACTGACCCCGGCGGGGCCC IGTTCTCCGTGTTGGTCCTGCAGGGGAGCGAAAACGACGGCCCCCAGCTTCACGCIGGCCAC CATCAAGGGGGACGAATACACCTTCACCTCCAGTAATGCTGAGGACATTCGTGACCIGGTGG TCAeCTTCCTAGAGGGGCTCCGGAAGAGATCTAAGTATGTTGTGGCCCTGCAGGATAACCCC AACCCCGCAGGCGAGGAGTCAGGCTTCCTCAGCITTGCCAAGGGAGACCTCATCATCCTGGA ccatgacacgggcgagcaggtcatgaactcgggctgggccaacggCatcaatgagaggacca agcagcGtggggacttccccaccgactGtgtgtacgtcatgcccactgtcaccatgccacct CGTGAGATTGTGGCCCTGGTGACCATGACTCCeGATCAGAGGCAGGACGTTGTCCGGCTCTT GCAGCTGGGAACGGCGGAGCGCGAGGTGCGTGGGAAGGCCTACACGCTGGAGGAGΤΤΤTCCT ATGACTACTTCAGGCCCCCAGCCAAGCAGACGCTGAGGCGTGICATGGTGTCCAAGGCCCGA GGCAAGGAC CGGCTGTGGAGCCACACGCGGGAACCGCT C AAGC AGGCGC T GC TCAAGAAGCI CCTGGGCAGTGAGGAGCT CTGGCAGGAGGCCT GCGT GGCC I1CATIGC TG TGCT C AAGTACA tgggggactaccggtccaagaggacacgctccgtcaatgagctcaccgaccagatctttgag GGICCeCTGAAAGCCGAGCCGCTGAAGGACGAGGCATATGTGCAGATCCIGAAGCAGCTGAe CGACAAeCACATCAGGTACAGCGAGGAGCGGGGTTGGGAGCTGCTCTGGCTGTGCACGGGCC TTTTCCCACCCAGCAACATCCTCCTGCCCCACGTGCAGCGCTTCCTGCAGTCCCGAAAGCAC tppppaptppppatppaptppptpp a αρρ/-.ρ,_,ρραγ'.α a α'-.γ-ρρτραρα a a^pppt'-ppppa a

G T ACC C T CCGCACCTGGTGGAGGT GGAGGCCATCCAGCACAAGACGACCCAGAT Τ Τ TCC AC A

AGG T C T ACT T CCC TGATGACACTGACGAGGCCTT CGAAGTGGAGTGCAGCACCAAGGCCAAG

GACTTCTGCCAGAACATCGCCACCAGGCTGCTCCTC'AAGTCCTCAGAGGGATTCAGCCTCTT

TGTCAAAATTGCAGACAAGGTCATCAGCGT TC C TGAGAAT GACT T C Τ T CT T T GAC T TT GT T C

GACACTTGACAGACTGGATAAAGAAAGCTCGGCCCATCAAGGACGGAATTGTGCCCTCACTC

ACCTACCAGGTGTTCTTCATGAAGAAGCTGTGGACCACCACGGTGCCAGGGAAGGATCCCAT

GGCCGATTCCATCTTCCACTATTACCAGGAGTTGCCCAAGTATCTCCGAGGCTACCACAAGT

GC AC GCGGGAGGAGGTGCTGCAGGTGGGGGCGCTGAT C TACAGGGT C AAGT ICGAGGAGGAC

AAGTCCTACTTCCCCAGCATCCCCAAGCTGCTGCGGGAGCTGGTGCCCCAGGACCTTATCCG

GCAGGTCTCAGCTGATGACTGGAAGeGGTCCATCGTCGCCTACTTCAACAAGCACGCAGGGA

AGTCCAAGGAGGAGGCCAAGCTGGCCTTCCTGAAGGTCATCTTCAAGTGGCCCAC CTTTGGC

TCAGCCTTCTTCGAGGTGAAGCAAACTACGGAGCCAAAGTTCCCTGAGATCCTCCTAATTGC

CATCAACAAGTATGGGGTCAGCCT CATCGATCCCAAAACGAAGGATATCCTGACCACTCAT C

CCTTCACCAAGATCTCCAACTGGAGCAGCGGCAACACCTACTTCCACATCACCATTGGGAAC

TTGGTGCGCGGGAGCAAACTGCTCTGCGAGACGTCACTGGGCTACAAGATGGATGACCTCCT

GACTTCCTACATTAGCCAGATGCTCACAGCCATGAGCAAACAGCGGGGCTCCAGGAGCGGCA

AGTGACCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGAGATCTGCCTCGACT

GTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCeCGTGCCTTCCTTGACCCTGGA

AGG T GC CAC T CCC AC T GT CCTT TCC TAATAAAAT GAGGAAAT TGCATCGCATTGTC T GAG TA ggtgtcattctattCtgggGggtgggGtggggcaggacagCaagggGgaggattgggaagac aatagcaggcatgctggggactcgagttaagggcgcaattcccgattaggatcttcctagag catggctacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgat

GGAGTTGGGCACTCCCTCTCTGCGCGCTCGCTCGCTCAGTGAGGCGGGGCGACCAAAGGTCG CCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG (SEQ ID No. 28) AP: gtgatcctaggtggaggccgaaagtacatgtttcgcatgggaaccccAgaccctgagtaccc agatgactacagccaaggtgggaccaggctggacgggaagaatctggtgcaggaatggctgg

CGAAGCGCCAGGGTGCGCGGTACGTGTGGAACCGCACTGAGCTCATGCAGGCTTCCCTGGAC

CCGTCTGT GAC CCAT CTCAT GGGT CT CTTT GAGC CTGGAGACAT GAAATAC GAGAT CCACC G AG ACT C CAC ACT GG AGCC CT CCGT GAT GGA (SEQ ID No. 29) 3XFLAG TAG: gActacAaagaccatGacGgtgattataaagatcatgacaccgactacaagGatgacgatga GAAG (SEQ ID No. 30) HA: ATGTATGATGTTCCTGATTATGCTAGCCTC (SEQ ID No. 31) [0070] For the purposes of this invention, a coding sequence of ABCA4, MYO7Aand CEP290 which are preferably respectively selected from the sequences herein enclosed, or sequences encoding the same amino acid sequence due to the degeneracy of the genetic code, is functionally linked to a promoter sequence able to regulate the expression thereof in a mammalian retinal cell, particularly in photoreceptor cells. Suitable promoters that can be used according to the invention include the cytomegalovirus promoter, Rhodopsin promoter, Rhodopsin kinase promoter, Interphotoreceptor retinoid binding protein promoter, vitelliform macular dystrophy 2 promoter, fragments and variants thereof retaining a transcription promoter activity.

[0071] Viral delivery systems include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectors, pseudotyped AAV vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors. Pseudotyped AAV vectors are those which contain the genome of one AAV serotype in the capsid of a second AAV serotype; for example an AAV2/8 vector contains the AAV8 capsid and the AAV 2 genome (Auricchio et al. (2001) Hum. Mol. Genet. 10(26):3075-81). Such vectors are also known as chimeric vectors. Other examples of delivery systems include ex vivo delivery systems, which include but are not limited to DNA transfection methods such as electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection.

[0072] The construction of an AAV vector can be carried out following procedures and using techniques which are known to a person skilled in the art. The theory and practice for adeno-associated viral vector construction and use in therapy are illustrated in several scientific and patent publications (the following bibliography is herein incorporated by reference: Flotte TR. Adeno-associated virus-based gene therapy for inherited disorders. Pediatr Res. 2005 Dec;58(6):1143-7; Goncalves MA. Adeno-associated virus: from defective virus to effective vector, Virol J. 2005 May 6;2:43; Surace EM, Auricchio A. Adeno-associated viral vectors for retinal gene transfer. Prog Retin Eye Res. 2003 Nov;22(6):705-19; Mandel RJ, Manfredsson FP, Foust KD, Rising A, Reimsnider S, Nash K, Burger C. Recombinant adeno-associated viral vectors as therapeutic agents to treat neurological disorders. Mol Ther. 2006 Mar; 13(3):463-83).

[0073] Suitable administration forms of a pharmaceutical composition containing AAV vectors include, but are not limited to, injectable solutions or suspensions, eye lotions and ophthalmic ointment. In a preferred embodiment, the AAV vector is administered by subretinal injection, e.g. by injection in the subretinal space, in the anterior chamber or in the retrobulbar space. Preferably the viral vectors are delivered via subretinal approach (as described in Bennicelli J, et al Mol Ther. 2008 Jan 22; Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer).

[0074] The doses of virus for use in therapy shall be determined on a case by case basis, depending on the administration route, the severity of the disease, the general conditions of the patients, and other clinical parameters. In general, suitable dosages will vary from 108 to 1013 vg (vector genomes)/eye. AAV vector production [0075] AAV vectors were produced by the TIGEM AAV Vector Core by triple transfection of HEK293 cells followed by two rounds of CsC12 purification (54). For each viral preparation, physical titers [genome copies (GC)/mlj were determined by averaging the titer achieved by dot-blot analysis (55) and by PCR quantification using TaqMan (54) (Applied Biosystems, Carlsbad, CA). AAV infection of HEK293 cells [0076] HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 2 mM L-glutamine (GIBCO, Invitrogen S.R.L., Milan,

Italy). Cells were plated in six-well plates at a density of 2χ106 cells/well and transfected 16 hours later with 1,3 pg of pDeltaF6 helper plasmid which contains the Ad helper genes (56) using the calcium phosphate method. After 5 hours, cells were washed once with DMEM and incubated with AAV2/2 vectors (m.o.i: 105 GC/cell of each vector; 1:1 co-infection with dual AAV vectors resulted in of 2x105 total GC/cell) in a final volume of 700 pL serum-free DMEM. Two hours later 2 ml of complete DMEM was added to the cells. Cells were harvested 72 hours following infection for Western blot analysis.

Animal models [0077] This study was carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals, the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and the Italian Ministry of Health regulation for animal procedures. Mice were housed at the Institute of Genetics and Biophysics animal house (Naples, Italy) and maintained under a 12-hour light/dark cycle (10-50 lux exposure during the light phase). C57BL/6 and BALB/c mice were purchased from Harlan Italy SRL (Udine, Italy). Albino Abca4-/- mice were generated through successive crosses and backcrosses with BALB/c mice (homozygous for Rpe65 Leu450) (57) and maintained inbred. Breeding was performed crossing homozygous mice. Pigmented sh14626SB/4626SB (referred to as sh1-/-) mice were imported from the Wellcome Trust Sanger Institute (Cambridge, UK, a kind gift of Dr. Karen Steel) and back-crossed twice with CBA/Ca mice purchased from Harlan Italy SRL (Udine, Italy) to obtain heterozygous sh1+/4626SB (referred to as sh1+/-) mice to expand the colony. The mice were maintained intercrossed; breeding was performed crossing heterozygous females with heterozygous males. The pigmented sh1 mice used in this study were either Usher 1B affected (sh1-/-) or unaffected (sh1+/- and sh1+/+). The genotype for the MYO7A4626SB allele was performed by PCR analysis of genomic DNA (extracted from the mouse tail tip) followed by DNA sequencing. The primers used for the PCR amplification are as follows: Fw1 (GTGGAGCTTGACATCTACTTGACC) and Rev3 (AGCTGACCCTCATGACTCTGC), which generate a product of 712 bp that was sequenced with the Fw1 primer. The Large White Female pigs used in this study were registered as purebred in the LWHerd Book of the Italian National Pig Breeders' Association (Azienda Agricola Pasotti, Imola, Italy).

Subretinal injection of AAV vectors in mice and pigs [0078] Mice (4-5 weeks-old) were anesthetized with an intraperitoneal injection of 2 ml/100 g body weight of avertin [1.25% w/v of 2,2,2-tribromoethanol and 2.5% v/v of 2-methyl-2-butanol (Sigma-Aldrich, Milan, Italy)] (58), then AAV2/8 vectors were delivered subretinally via a trans-scleral transchoroidal approach as described by Liang et al (59). All eyes were treated with 1 pL of vector solution. The AAV2/8 doses (GC/eye) delivered vary across the different mouse experiments as it is described in the "RESULTS" section. AAV2/1-CM\/-human Tyrosinase (60) (dose: 2x108 GC/eye) or AAV2/5-CMV-EGFP (encoding normal size EGFP, dose: 4x108 GC/eye) was added to the AAV2/8 vector solution that was subretinally delivered to albino (Abca4-I- and BALB/c) (Fig. 6B, 7-8) or pigmented sh1 mice (Fig. 10-11), respectively. This allowed us to mark the RPE within the transduced part of the eyecup, which was subsequently dissected and analyzed. (Fig. 6B, 7-8, 10-11). Subretinal delivery of AAV vectors to the pig retina was performed as previously described (11). All eyes were treated with 100 pL of AAV2/8 vector solution. The AAV2/8 dose was 1x1010 (Fig. 3B) or 1x1011 GC of each vector/eye (Fig. 5B and 16) and co-injection of dual AAV vectors resulted in a total dose of 2x1010 GC/eye or 2x1011 GC/eye, respectively.

Western blot analysis [0079] Samples (HEK293 cells, retinas or eyecups) for Western blot analysis were lysed in RIPA buffer (50 mM Tris-Hcl pH 8.0, 150mM NaCI, 1% NP40, 0.5% Na-Deoxycholate, 1mM EDTA pH 8.0, 0.1% SDS) to extract EGFP and MYO7A proteins, or in SIE buffer (250 mM sucrose, 3 mM imidazole pH 7.4, 1% ethanol, and 1% NP-40) to extract ABCA4 protein.

[0080] Pig samples (the treated areas of the retina as well as whole RPE sheets) were lysed in RIPA buffer to extract MYO7A from RPE sheets, and in SIE buffer to extract MYO7A and ABCA4 from retinas.

[0081] Lysis buffers were supplemented with protease inhibitors (Complete Protease inhibitor cocktail tablets, Roche, Milan, Italy) and 1 mM phenylmethylsulfonyl. After lysis EGFP and MYO7A samples were denatured at 99°C for 5 minutes in IX Laemli Sample buffer; ABCA4 samples were denatured at 37°C for 15 minutes in IX Laemli sample buffer supplemented with 4M urea. Lysates were separated by 7% (ABCA4 and MYO7A samples) or 12% (EGFP samples) SDS-polyacrylamide gel electrophoresis. The antibodies used for immuno-blotting are as follows: anti EGFP (sc-8334, Santa Cruz, Dallas, Texas, USA, 1:500); anti-3xflag (A8592, Sigma-Aldrich, 1:1000); anti-Myo7a (polyclonal, Primm Sri, Milan, Italy, 1:500) generated using a peptide corresponding to aminoacids 941-1070 of the human MYO7A protein; anti-HA antibody (PRB-101 P-200, HA.11, Covance, Princeton, NJ, USA, 1:2000); anti-β Tubulin (T5201,Sigma Aldrich, 1:10000); anti-Filamin A (catalog#4762, Cell Signaling Technology, Danvers, MA, USA, 1:1000); anti-Dysferlin (Dysferlin, clone Ham1/7B6, MONX10795, Tebu-bio, Le Perray-en-Yveline, France, 1:500). The quantification of EGFP, ABCA4 and MYO7A bands detected by Western blot was performed using ImageJ software (free download is available at http://rsbweb.nih.gov/ij/). ABCA4 and MYO7A expression was normalized to Filamin A or Dysferlin for the in vitro and in vivo experiments, respectively. EGFP expression was normalized to β-Tubulin or pg of proteins for in vitro and in vivo experiments, respectively. Different proteins were used for normalization based on the similarity of their molecular weight to those of the different transgene products.

Fundus photography [0082] The fundus live-imaging was performed by dilating the eye of C57BL/6 with a drop of tropicamide 1% (Visufarma, Rome, Italy) and subsequent eye stimulation with a 300W flash. Fundus photographs were taken using a Topcon TRC-50IX retinal camera connected to a charge-coupled-device Nikon D1H digital camera (Topcon Medical System, Oakland, NJ, USA).

Histology, light and fluorescence microscopy [0083] To evaluate EGFP expression in histological sections, eyes from C57BL/6 mice or Large White pigs (11) were enucleated one month after AAV2/8 injection. Mouse eyes were fixed in 4% paraformaldehyde over-night and infiltrated with 30% sucrose over-night; the cornea and the lens were then dissected and the eyecups were embedded in optimal cutting temperature compound (O.C.T. matrix, Kaltek, Padua, Italy). Pig eyes were fixed in 4% paraformaldehyde for 48 hours, infiltrated with 10% sucrose for 4 hours, 20% sucrose for 4 hours and finally 30% sucrose overnight. Then, the cornea, the lens, and the vitreous body were dissected and the EGFP-positive portions of the eyecups were embedded in optimal cutting temperature compound (O.C.T. matrix, Kaltek). Serial cryosections (10 pm thick) were cut along the horizontal meridian and progressively distributed on slides. Retinal histology pictures were captured using a Zeiss Axiocam (Carl Zeiss, Oberkochen, Germany). To analyze melanosome localization in the RPE of pigmented sh1 mice, eyes were enucleated 2 months following the AAV injection, fixed in 2% glutaraldehyde-2% paraformaldehyde in 0.1 M phosphate buffer over-night, rinsed in 0.1 M phosphate buffer, and dissected under a florescence microscope. The EGFP-positive portions of the eyecups were embedded in Araldite 502/EMbed 812 (catalog #13940, Araldite 502/EMbed 812 KIT, Electron Microscopy Sciences, Hatfield, PA, USA). Semi-thin (0.5-pm) sections were transversally cut on a Leica Ultratome RM2235 (Leica Microsystems, Bannockburn, IL, USA), mounted on slides, and stained with Epoxy tissue stain (catalog #14950, Electron Microscopy Sciences). Melanosomes were counted by a masked operator analyzing 10 different fields/eye under a light microscope at 100X magnification. Retinal pictures were captured using a Zeiss Axiocam (Carl Zeiss).

Electron microscopy and immuno-gold labelling [0084] For electron microscopy analyses eyes were harvested from Abca4-/- or sh1 mice at 3 and 2 months after AAV injection, respectively. Eyes were fixed in 0.2% glutaraldehyde-2% paraformaldehyde in 0.1 Μ PHEM buffer pH 6.9 (240 mM PIPES, 100 mM HEPES, 8mM MgCI2, 40 mM EGTA) for 2 hours and then rinsed in 0.1 M PHEM buffer. Eyes were then dissected under light or fluorescence microscope to select the Tyrosinase- or EGFP-positive portions of the eyecups of albino (Abca4-I- and BALB/c) and pigmented sh1 mice, respectively. The transduced portion of the eyecups were subsequently embedded in 12% gelatin, infused with 2.3M sucrose and frozen in liquid nitrogen. Cryosections (50 nm) were cut using a Leica Ultramicrotome EM FC7 (Leica Microsystems) and extreme care was taken to align PR connecting cilia longitudinally. Measurements of RPE thickness and counts of lipofuscin granules in Abca4-I- eyes were performed by a masked operator (Roman Polishchuk) using the iTEM software (Olympus SYS, Hamburg, Germany). Briefly, RPE thickness was measured in at least 30 different areas along the specimen length using the "Arbitrary Line" tool of iTEM software. The "Touch count" module of the iTEM software was utilized to count the number of lipofuscin granules in the 25pm2 areas distributed randomly across the RPE layer. The granule density was expressed as number of granules per 25pm2. The immuno-gold analysis aimed at testing the expression of ABCA4-HA in Abca4-I- samples after AAV vector delivery was performed by incubating cryosections successively with monoclonal anti-HA antibody (MMS-101 P-50, Covance, 1:50), rabbit anti-mouse IgG, and 10-nm gold particle-conjugated protein A. To quantify rhodopsin localization to the connecting cilium of sh1 PR, cryosections of sh1 mice were successively incubated with anti-rhodopsin antibody (1D4, ab5417, Abeam, Cambridge, UK, 1:100), rabbit anti-mouse IgG, and 10-nm gold particle-conjugated protein A. The quantification of gold density of rhodospin in the connecting cilia was performed by a masked operator using iTEM software (Olympus SYS). Briefly, the "Touch count" module of the iTEM software was used to count the number of gold particles per cilium that were normalized to the cilium perimeter (nm) that was measured using the "Closed polygon tool". Gold density was expressed as gold particles/nm. Immunogold labelled cryosections were analyzed under FEI Tecnai-12 (FEI, Eindhoven, The Netherlands) electron microscope equipped with a Veletta CCD camera for digital image acquisition.

Electrophysiological analyses [0085] To assess the recovery from light desensitization eyes were stimulated with 3 light flashes of 1 cd s/m2 and then desensitized by exposure to constant light (300 cd/m2) for 3 minutes. Then, eyes were stimulated over time using the pre-desensitization flash (1 cd s/m2) at 0, 5, 15, 30, 45 and 60 minutes post-desensitization. The recovery of rod activity was evaluated by performing the ratio between the b-wave generated post-desensitization (at the different time points) and that generated pre-desensitization. The recovery from light desensitization was evaluated in 2-month-old Abca4-/- mice at 6 weeks post treatment (Fig. 13).

Statistical analysis [0086] Data are presented as meant standard error of the mean (s.e.m.). Statistical p values <0.05 were considered significant. One-way ANOVA with post-hoc Multiple Comparison

Procedure was used to compare data depicted in: Figure 2 (p ANOVA: A. 0.0002; B. 0.0015; C. 2x1 O'7); Figure 8B (pANOVA: 0.076); Figure 11B (pANOVA: 0.5). As lipofuscin granules (Fig. 7B) and melanosomes (Fig. 10B) were counted, counts were analyzed by deviance from a Negative Binomial generalized linear models (61) (Fig. 7B: p value analysis of deviance 0.03794; Fig. 10B: p value analysis of deviance «2x10"10). The statistically significant differences between groups determined with the post-hoc Multiple Comparison Procedure are marked by asterisks in the Figures.

RESULTS

[0087] Generation of normal size, oversize and dual AAV vectors.

[0088] The inventors generated oversize (OZ), dual AAV trans-splicing (TS), and hybrid vectors that included either the reporter EGFP, the therapeutic ABCA4-3xflag or the MYO7A-HA coding sequences. The inventors also generated dual AAV trans-splicing (TS), and hybrid vectors that included the therapeutic CEP290 tagged at its C-terminus with HA tag. The recombinogenic sequences included in the dual AAV hybrid vectors were based on either a previously reported region of the alkaline phosphatase transgene (AP, dual AAV hybrid AP) (39) or a 77 bp sequence from the the F1 phage genome (AK, dual AAV hybrid AK) that the inventors found to be recombinogenic in previous experiments (Colella and Auricchio, unpublished data). The inventors also generated dual AAV overlapping (OV) vectors for ABCA4, MY07A and CEP290. The inventors did not generate dual AAV OV vectors for EGFP because the efficiency of this approach relies on transgene-specific overlaps for reconstitution (38) and therefore cannot be extrapolated from one gene to another. Instead, for EGFP the inventors generated single AAV vectors of normal size (NS) to compare levels of transgene expression from the various strategies. The constructs generated for production of all AAV vectors used in this study are listed in Table 1 and a schematic representation of the various approaches is depicted in Figure 1.

[0089] The inventors used AAV2/2 vectors for the in vitro experiments, with the ubiquitous cytomegalovirus (CMV) or chicken beta-actin (CBA) promoters, which efficiently transduce HEK293 cells (40). In addition, since the use of heterologous ITRs from AAV serotypes 2 and 5 can increase the productive reassembly of dual AAV vectors (51), the inventors also generated dual AAV AK vectors with heterologous ITRs (Fig. 17a) encoding ABCA4 and MY07A. AAM vectors with heterologous ITRs were packaged in AAV capsids from serotype 2 and tested in vitro.

[0090] In the experiments performed in vivo in the retina, The inventors used AAV2/8 vectors, which efficiently transduce RPE and PR (10-12) but poorly infect HEK293 cells, and either the ubiquitous CBA and CMV promoters (11), or the RPE-specific vitelliform macular dystrophy 2 (VMD2) (41) or the PR-specific Rhodopsin (RHO) and Rhodopsin kinase (RHOK) promoters (10) (Table 1).

[0091] Dual AAV vectors allow high levels of transduction in vitro.

[0092] The inventors initially compared the efficiency of the various OZ, dual AAV OV, TS and hybrid AP and AK strategies for AAV-mediated large gene transduction in vitro by infecting HEK293 cells with the AAV2/2 vectors [multiplicity of infection, m.o.i.: 105 genome copies (GC)/cell of each vector] with ubiquitous promoters (CMV for EGFP, ABCA4-3xflag, and CEP290-HA, and CBA for MYO7A-HA).

[0093] Cell lysates were analyzed by Western blot with anti-EGFP (Fig. 2A), -3xflag (to detect ABCA4-3xflag, Fig. 2B), -MYO7A(Fig. 2C) and -HA (to detect CEP290-HA) (Fig. 12A) antibodies. Representative Western blots are shown in Figure 2A-C and 12A. All strategies resulted in the expression of proteins of the expected size. As predicted, no bands of the expected size were observed when only one of the dual AAV vectors was used for infection (Fig. 2A-C and 12A). Quantification of transgene expression (Figure 2D-F) showed that the dual AAV hybrid AP approach resulted in the lowest levels of transgene expression, while the dual AAV OV, TS and hybrid AK approaches were more efficient than the AAV OZ approach. Dual AAV TS and hybrid AK approaches confirmed their ability to efficiently express large genes also in the case of CEP290 (Fig. 12B). In addition, the use of dual AAV AK vectors with heterologous ITRs resulted in expression of full-length ABCA4 and MYO7A proteins in vitro (Fig. 17).

[0094] Dual AAV TS and hybrid AK but not OV vectors transduce mouse and pig photoreceptors.

[0095] The inventors then evaluated each of the AAV-based systems for large gene transduction in the mouse retina. To test the dual AAV OV, which was transgene-specific, The inventors used the therapeutic ABCA4 and MY07A genes (Fig. 3). The inventors used EGFP to evaluate the AAV OZ and the dual AAV TS, hybrid AP and AK approaches (Fig. 4). Western blot analysis on retinal lysates, one month after subretinal delivery in C57BL/6 mice of the dual AAV OV vectors (dose of each vector/eye: 1.3x109 GC), encoding ABCA4-3xflag from the ubiquitous CMV promoter, revealed robust protein expression (Fig. 3A). To determine which cell type in the retina expressed ABCA4, The inventors used dual AAV OV vectors that contained either the PR-specific RHO and RHOK, or the RPE-specific VMD2 (dose of each vector/eye: 1x109 GC) promoters. The inventors detected ABCA4 protein expression in retinas injected with the VMD2 but not in those containing the RHO and RHOK promoters (Fig. 3A). These results were also confirmed in the Large White pig retina. The pig retina is an excellent model to evaluate vector efficiency because of its size, which is similar to the human retina, and because it is enriched with cones that are concentrated in a streak-like region whose cone density is comparable to that of the primate macula (11). The inventors injected Large White pig subretinally with dual AAV OV vectors encoding ABCA4-3xflag (dose of each vector/eye: 1x101° GC), and observed ABCA4 protein expression with the CMV but not the RHO promoter (Fig. 3B). Similarly, subretinal administration of dual AAV OV vectors encoding MYO7A-HA resulted in weak MYO7A protein expression in the mouse retina with the ubiquitous CBA (dose of each vector/eye: 2.5x109 GC) and no detectable expression with the RHO (dose of each vector/eye: 3.2x109 GC) promoter (Fig. 3C). Overall, these data suggested that the dual AAV OV approach was more efficient for large gene transfer to RPE than to PR, which are a major target of gene therapy for IRDs, such as STGD and USH1B.

[0096] To find an AAV-based strategy that efficiently transduces large genes in PR, the inventors evaluated the retinal transduction properties of the AAV OZ and dual AAV TS, hybrid AP, and AK approaches. The inventors initially used EGFP, which allowed us to easily localize transgene expression in the various retinal cell types including PR as well as to properly compare the levels of AAV-based large transgene transduction to those of a single AAV NS vector. C57BL/6 mice were subretinally injected with AAV NS, OZ and dual AAV TS, and hybrid AP and AK vectors (dose of each vector/eye: 1.7x109 GC), all encoding EGFP under the transcriptional control of the CMV promoter. One month later, fundus photographs showed that the highest levels of fluorescence were obtained with the AAV NS, and dual AAV TS and hybrid AK approaches (Fig. 15). Fluorescence microscope analysis of retinal cryosections showed that detectable levels of RPE or PR transduction could be observed in: 77% (10/13) retinas injected with AAV NS and OZ vectors; 92% (12/13) retinas injected with dual AAV TS, hybrid AP and AK vectors. Figure 4 shows the best transduced retinas from each of these groups. The most robust levels of PR transduction were obtained with the AAV NS and dual AAV TS and hybrid AK approaches.

[0097] The inventors then assessed PR-specific transduction levels in C57BL/6 mice following subretinal administration of dual AAV TS and hybrid AK vectors, which appears the most promising for large gene reconstitution in PR, as well as AAV NS vectors for comparison (dose

of each vector/eye: 2.4x109 GC). All vectors encoded EGFP under the transcriptional control of the PR-specific RHO promoter. One month after vector administration retinas were cryosectioned and analyzed under a fluorescence microscope (Fig. 5A). All approaches resulted in high levels of PR transduction, which seemed more consistent with the single AAV NS vector. The inventors found PR transduction in: 100% (6/6) of the retinas injected with AAV NS; 60% (9/15) of the retinas injected with dual AAV TS; 71% (10/14) of the retinas injected with dual AAV hybrid AK. Figure 5A shows the best transduced retinas from each of these groups. Thus, the inventors conclude that dual AAV TS and hybrid AK strategies allow efficient mouse PR transduction although at levels which are lower than those obtained with a NS AAV. The inventors then confirmed that subretinal administration of dual AAV TS and hybrid AK vectors (dose of each vector/eye: 1x1011GC; EGFP-positive retinas out of total injected: 2/2 dual AAV TS; 2/2 dual AAV hybrid AK) transduced PR of White Large pigs (Fig. 5B).

[0098] In addition, subretinal delivery to the pig retina of dual AAV TS and hybrid AK vectors (dose of each vector/eye: 1x1011) resulted in efficient expression of both full-length ABCA4-3xflag specifically in PRs (Fig. 16a) and full-length MYO7A-HA in RPE and PRs (Fig. 16b) Interestingly, dual AAV hybrid AK vectors resulted in more consistent expression of the large ABCA4 and MYO7A proteins in PRs, compared with dual AAV TS vectors (Fig. 16).

[0099] Dual AAV vectors improve the retinal phenotype of STGD and USH1B mouse models.

[0100] To understand whether the levels of PR transduction obtained with the dual AAV TS and hybrid AK approaches may be therapeutically relevant, the inventors investigated them in the retina of two mouse models of IRDs, STGD and USH1B caused by mutations in the large ABCA4 and MY07A genes, respectively.

[0101] Although the Abca4-i- mouse model does not undergo severe PR degeneration (42), the absence of the ABCA4-encoded all-trans retinal transporter in PR outer segments (43-44) causes an accumulation of lipofuscin in PR as well as in RPE, as result of PR phagocytosis by RPE (45). As a consequence, both the number of lipofuscin granules in the RPE and the thickness of RPE cells are greater in Abca4-i- mice than in control mice (45). Moreover the Abca4-i- mouse model is characterized by delayed dark adaptation (57, 62). Since ABCA4 is expressed specifically in PR, the inventors generated dual AAV TS and hybrid AK vectors encoding ABCA4-3xflag under the transcriptional control of the RHO promoter. These vectors were subretinally injected in wild-type C57BL/6 mice (dose of each vector/eye: 3-5x109 GC) and one month later retinas were lysed and analyzed by Western blot with anti-3xflag antibodies. Both approaches resulted in robust yet variable levels of ABCA4-3xflag expression. ABCA4-3xflag expression levels were more consistent in retina treated with the dual AAV hybrid AK vectors (Fig. 6A). These results were confirmed in Large White pigs (data not shown). In addition, one month-old albino Abca4-i- mice were injected subretinally with the dual AAV hybrid AK RHO-ABCA4-HA vectors (dose of each vector/eye: 1-3x109 GC). Three months later, eyes were harvested and immuno-electron microscopy analysis with antihemagglutinin (HA) antibodies of retinal sections confirmed that immunogold particles were correctly localized in PR outer segments only in animals that were injected with the combination of 5' and 3' dual AAV hybrid AK vectors (Fig. 6B). To assess the functionality of the ABCA4 protein expressed by the dual vectors, the inventors also performed transmission electron microscopy to assess the presence and number of RPE lipofuscin granules (Fig. 7) and RPE thickness (Fig. 8). Both were greater in the retina of Abca4 -I- mice injected with control vectors than in the retina of wild-type, age-matched Balb/C controls, and were reduced or normalized in the eyes injected with the therapeutic dual AAV TS or hybrid AK vectors (Fig. 7B and 8B). In addition, the ability of Abca4-/- photoreceptors to recover from light desensitization was significantly improved in the retinas treated with the therapeutic vectors when compared to control retinas (Fig. 13).

[0102] The inventors then tested PR transduction levels and efficacy of dual AAV-mediated MY07A gene transfer in the retina of sh1 mice, the most commonly used model of USH1B (23-24, 46-48). In sh1 mice, a deficiency in the motor Myo7a causes the mis-localization of RPE melanosomes (47), which do not enter into the RPE microvilli, and the accumulation of rhodopsin at the PR connecting cilium (48). Since MY07A is expressed in both RPE and PR (22-23), the inventors then used dual AAV TS and hybrid AK vectors expressing MYO7AHA under the transcriptional control of the ubiquitous CBA promoter. One month-old wild-type C57BL/6 mice were injected with the dual AAV vectors (dose of each vector/eye: 1.7x109 GC) and eyecup lysates were evaluated one month later using Western blot analysis with anti-HA antibodies. Results showed similarly robust and consistent levels of MY07A expression in retinas treated with both approaches (Fig. 9). Taking advantage of our anti-MYO7A antibody able to recognize both murine and human MY07A, we compared the levels of MY07A achieved following delivery of dual AAV vectors to the sh1-/- eye to those expressed endogenously in the sh1+/+ eye (Fig. 14). We used both the CBA (Fig. 14, left panel, dose of each vector/eye: 1-6x109GC) and the RHO promoters (Fig. 14, right panel, dose of each vector/eye: 2x109GC) to distinguish MY07A expression achieved in both PR and RPE from that in PR alone: the former is about 20% (Fig. 14, left panel) and the latter up to about 50% of endogenous Myo7a (Fig. 14, right panel). Our analysis additionally shows that the levels of MYO7A expression achieved in PR by dual AAV hybrid AK are higher than those obtained with the dual AAV TS vectors despite the number of transduced retinas is similar (TS-MYO7A: 3 retinas positive out of 8 injected; AK-MY07A: 4 retinas positive out of 8 treated; Fig. 14, right panel).

[0103] To test the ability of MYO7A expressed from dual AAV vectors to rescue the defects of the sh1-/- retina, the inventors then subretinally injected the CBA sets of dual AAV TS and hybrid AK vectors (dose of each vector/eye: 2.5x109 GC) in one month-old s/?1 mice. The inventors assessed RPE melanosome (Fig. 10) and rhodopsin localization (Fig. 11) by analysis of semi-thin retinal section and by immuno-electron microscopy, respectively. Unlike unaffected sh1+/-, the sh1-/- melanosomes do not enter the RPE microvilli after delivery of control vectors (each single 5' half of the dual-AAV strategies, Fig. 10). The number of RPE melanosomes correctly localized apically was significantly improved after the delivery of either dual AAV TS or hybrid AK vectors encoding MY07A (Fig. 10B). Remarkably, the inventors also found that the MYO7A expression mediated by dual AAV TS and hybrid AK vectors reduced the accumulation of rhodopsin at the connecting cilium of sh1-/- PR (Fig. 11).

DISCUSSION

[0104] While AAV-mediated gene therapy is effective in animal models and in patients with inherited blinding conditions (5-9, 49), its application to diseases affecting the retina and requiring a transfer of genes larger than 5kb (referred to as large genes) is inhibited by AAV limited cargo capacity. To overcome this, the inventors compared the efficiency of various AAV-based strategies for large gene transduction including: AAV OZ and dual AAV OV, TS and hybrid approaches in vitro and in mouse and pig retina. In previous experiments, inventors selected a 77 bp sequence from the F1 phage genome that the inventors identified for its recombinogenic properties and used in the dual hybrid approach (AK, dual AAV hybrid AK).

[0105] The inventors' in vitro and in vivo results show that the dual AAV hybrid AK surprisingly outperforms the dual AAV hybrid AP and that all dual AAV strategies the inventors tested (with the exception of the dual AAV hybrid AP) outperform AAV OZ vectors in terms of transduction levels. This may be explained by the homogenous size of the dual AAV genome population when compared to OZ genomes, which may favor the generation of transcriptionally active large transgene expression cassettes.

[0106] The dual AAV OV approach seems particularly interesting when compared to the TS or hybrid AK approaches as dual AAV OV vectors only contain sequences belonging to the therapeutic transgene expression cassette. However, when the inventors administered dual AAV OV vectors to the subretinal space of adult mice and pigs, the inventors were only able to detect expression of the large ABCA4 protein when the ubiquitous or the RPE-specific promoters, but not the PR-specific promoters, were used. This may suggest that the homologous recombination required for dual AAV OV reconstitution is more efficient in RPE than PR. This is consistent with the low levels of homologous recombination reported in postmitotic neurons (50) and may partially explain the lack of dual AAV OV-mediated MYO7A transduction recently reported by other groups (30). The inventors conclude that subretinal administration of dual AAV OV vectors should not be used for large gene transfer to PR, although the inventors cannot exclude that sequences that are more recombinogenic than those included in the inventors' dual AAV OV ABCA4 and MY07A vectors may allow efficient homologous recombination in PR.

[0107] Dual AAV TS and hybrid AK approaches efficiently transduce mouse and pig PR, differently from what the inventors observed with dual AAV OV. This is consistent with the knowledge that the mechanism of large gene reconstitution mediated by dual AAV TS and hybrid AK approaches may be via ITR-mediated head-to-tail rejoining (32, 35, 51) rather than homologous recombination.

[0108] The levels of mouse PR transduction the inventors achieved with dual AAV TS and hybrid AK is lower and less consistent than with single NS vectors. However, dual AAV may be effective for treating inherited blinding conditions that require relatively low levels of transgene expression, i.e. diseases inherited as autosomal recessive. Indeed, the inventors show that subretinal delivery of dual AAV TS and hybrid AK improves and even normalizes the retinal defects of two animal models of inherited retinal diseases, STGD and USH1B, which are due to mutations in large genes and are attractive targets of gene therapy.

[0109] The genome size of dual AAV vectors is homogenous, which means identity and safety issues related to their use should be less considerable than those related to AAV OZ vectors, which have heterogeneous genome sizes. In contrast, the inventors detected neither ERG or retinal histological abnormalities in the mice that the inventors followed up to 1-2 months after dual AAV vector delivery (data not shown).

[0110] In conclusion, the inventors identified a new recombinogenic sequence (AK) that strikingly improves the performance of the AAV dual hybrid vector system. In fact they found that dual AAV vectors are efficient both in vitro and in the retina in vivo. While dual AAV OV vectors efficiently transduce RPE, they do not transduce PR, whereas dual AAVTS and hybrid AK approaches drive efficient large gene reconstitution in both cell types. Administration of dual AAV TS and hybrid AK approaches improved the retinal phenotype of mouse models of STGD and USH1B, providing evidence of the efficacy of these strategies for gene therapy for these and other blinding conditions, which require large gene transfer to retinal PR as well as RPE. These findings will greatly broaden the application of AAV vectors for gene therapies not only to eyes, but also to muscle as well as to other organs and tissues. Diseases other than IRD caused by defective genes larger than 5 kb include non-limiting examples of muscular dystrophies, dysferlin deficencies (limb-girdle muscular dystrophy type 2B and Miyoshi myopathy), Cystic Fibrosis, Hemophilia.

REFERENCES

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SEQUENCE LISTING

[0112]

<110> FONDAZIONE TELETHON <120> Effective delivery of large genes by dual AAV vectors <130> PCT 122889 <150> US61/813,342 <151> 2013-04-18 <160>31 <170> Patentln version 3.5 <210> 1 <211> 82

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 1 gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga 60 cagagaagac tcttgcgttt ct 82 <210>2 <211> 51

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400>2 gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca g 51 <210> 3 <211 > 77

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400>3 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 60 gcgaatttta acaaaat 77 <210>4 <211> 130

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 4 ctgcgcgctc gctcgctCaC tgaggccgcc Cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgccegg ccteagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct 130 <210> 5 <211> 175

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 5 ctctcccccc tgtcgcgttc gctcgctcgc tggctcgttt gggggggtgg cagctcaaag 60 agctgccaga cgacggocct ctggccgtcg cccccccaaa cgagccagcg agcgagcgaa 120 cgcgacaggg gggagagtgc cacactctca agcaaggggg ttttgtaagc agtga 175 <210> 6 <211> 153

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400>6 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120 aatatgaccg ccatgttggc attgattatt gac 153 <21O> 7 <211 > 583

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400>7 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240 aagtccgcco cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300 catgacctta cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360 catggtgatg cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg 420 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480 ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt 540 aoggtgggag gtctatataa gcagagctcg tttagtgaac cgt 583 <210> 8 <211> 133

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400>8 gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga 60 cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc 120 tttctctcca cag 133 <210> 9 <211 > 2918

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400>9 atgggcttcg tgagacagat acagcttttg ctctggaaga actggaccct gcggaaaagg 60 caaaagattc gctttgtggt ggaactcgtg tggcctttat ctttatttct ggtcttgatc 120 tggttaagga atgccaaccc gctctacagc catcatgaat gccatttccc caacaaggcg 180 atgccctcag caggaatgct gccgtggctc caggggatct tctgcaatgt gaacaatccc 240 tgttttcaaa gccccacccc aggagaatct cctggaattg tgtcaaacta taacaactcc 300 atcttggcaa gggtatatcg agattttcaa gaactcctca tgaatgcacc agagagccag 360 caccttggcc gtatttggac agagctacac atcttgtccc aattcatgga caccctccgg 420 actcacccgg agagaattgc aggaagagga attcgaataa gggatatctt gaaagatgaa 480 gaaacactga cactatttct cattaaaaac atcggcctgt ctgactcagt ggtctacctt 540 ctgatcaact ctcaagtccg tccagagcag ttcgctcatg gagtcccgga cctggcgctg 600 aaggacatcg cctgcagcga ggccctcctg gagcgcttca tcatcttcag ccagagacgc 660 ggggcaaaga cggtgcgcta tgccctgtgc tccctctCcc agggcaccct acagtggata 720 gaagacactc tgtatgccaa cgtggacttc ttcaagctct tccgtgtgct tcccacactc 780 ctagacagcc gttctcaagg tatcaatctg agatcttggg gaggaatatt atctgatatg 840 tcaccaagaa ttcaagagtt tatccatcgg ccgagtatgc aggacttgct gtgggtgacc 900 aggCccctca tgcagaatgg tggtccagag acctttacaa agctgatggg catcCtgtct 960 gacctcctgt gtggctaccc cgagggaggt ggctctcggg tgctctcctt caactggtat 1020 gaagacaata actataaggc ctttctgggg attgactcca caaggaagga tcctatctat 1080 tcttatgaca gaagaacaac atccttttgt aatgcattga tccagagcct ggagtcaaat 1140 cctttaacca aaatcgcttg gagggcggca aagcctttgc tgatgggaaa aatcctgtac 1200 actcctgatt cacctgcagc acgaaggata ctgaagaatg ccaaCtCaac ttttgaagaa 1260 ctggaacacg ttaggaagtt ggtcaaagcc tgggaagaag tagggcccca gatctggtac 1320 ttctttgaca acagcacaca gatgaacatg atcagagata ccctggggaa cccaacagta 1380 aaagactttt tgaataggca gcttggtgaa gaaggtatta ctgctgaagc catcctaaac 1440 ttcctctaca agggccctcg ggaaagccag gctgacgaca tggccaactt cgactggagg 1500 gacatattta acatcactga tcgcaccctc cgccttgtca atcaatacct ggagtgcttg 1560 gtcctggata agtttgaaag ctacaatgat gaaactcagc tcacccaacg tgccctctct 1620 ctactggagg aaaacatgtt ctgggccgga gtggtattcc ctgacatgta tccctggacc 1680 agctctctac caccccacgt gaagtataag atccgaatgg acatagacgt ggtggagaaa 1740 accaataaga ttaaagacag gtattgggat tctggtccca gagctgatcc cgtggaagat 1800 ttccggtaca tctggggcgg gtttgcctat ctgCaggaca tggttgaaca ggggatcaca 1860 aggagccagg tgcaggcgga ggctccagtt ggaatctacc tccagcagat gccctacccc 1920 tgcttcgtgg acgattcttt catgatcatc ctgaaccgct gtttccctat cttcatggtg 1980 ctggcatgga tctactctgt ctccatgact gtgaagagca tcgtcttgga gaaggagttg 2040 cgactgaagg agaccttgaa aaatcagggt gtctccaatg cagtgatttg gtgtaqctgg 2100 ttcctggaca gcttctccat catgtcgatg agcatcttcc tcctgacgat attcatcatg 2160 catggaagaa tcctacatta cagcgaccca ttcatectct tcctgttctt gttggctttc 2220 tccactgcca ccatcatgct gtgctttctg ctcagcacct tcttctccaa ggccagtctg 2280 gcagcagcct gtagtggtgt catctatttc accctctacc tgccacacat cctgtgcttc 2340 gcctggcagg accgcatgac cgctgagctg aagaaggctg tgagcttact gtctccggtg 2400 gcatttggat ttggcactga gtacctggtt cgctttgaag agcaaggcct ggggctgcag 2460 tggagcaaca tcgggaacag tcccacggaa ggggacgaat tcagcttcct gctgtccatg 2520 cagatgatgc tccttgatgc tgctgtctat ggcttactcg cttggtacct tgatcaggtg 2580 tttccaggag actatggaac cccacttcct tggtactttc ttctacaaga gtcgtattgg 2640 cttggcggtg aagggtgttc aaccagagaa gaaagagccc tggaaaagac cgagccccta 2700 acagaggaaa cggaggatcc agagcaccca gaaggaatac acgactcctt ctttgaacgt 2760 gagcatccag ggtgggttcc tggggtatgc gtgaagaatc tggtaaagat ttttgagccc 2820 tgtggccggc cagctgtgga ccgtctgaac atcaccttct acgagaacca gatcaccgca 2880 ttcctgggcc acaatggagc tgggaaaacc accacctt 2918 <210> 10 <211> 130

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 10 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120 gagcgcgcag 130 <210> 11 <211 >4540

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 11 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgccegg ccteagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatetaegta gccatgctct 180 aggaagatet tcaatattgg ccattagcca tattattcat tggttatata gcataaatca 240 atattggcta ttggccattg catacgttgt atctatatca taatatgtac atttatattg 300 gctcatgtcc aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat 360 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg 420 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt 480 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac 540 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg 600 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact 660 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt 720 ggcagtacac caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc 780 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc 840 gtaataaccc cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 900 taagcagagc tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc 960 acagttaaat tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca 1020 gaagttggtc gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag 1080 accaatagaa actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta 1140 ttggtcttac tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt 1200 acagctctta aggctagagt acttaatacg actcactata ggctagcctc gagaattCac 1260 gcgtggtacc tctagagtqg acccgggcgg ccgccatggg cttcgtgaga cagatacagc 1320 ttttgctctg gaagaactgg accctgcgga aaaggcaaaa gattcgcttt gtggtggaac 1380 tcgtgtggcc tttatcttta tttctggtct tgatctggtt aaggaatgcc aaccegctct 1440 acagccatca tgaatgccat ttccccaaca aggcgatgcc ctcagcagga atgctgccgt 1500 ggctccaggg gatcttctgc aatgtgaaca atccctgttt tcaaagcccc accccaggag 1560 aatctcctgg aattgtgtca aactataaca actccatctt ggcaagggta tatcgagatt 1620 ttcaagaact cctcatgaat gcaccagaga gccagcacct tggccgtatt tggacagagc 1680 tacacatctt gtcccaattc atggacaccc tccggactca cccggagaga attgcaggaa 1740 gaggaattcg aataagggat atettgaaag atgaagaaac actgacacta tttctcatta 1800 aaaacatcgg cctgtctgac toagtggtct accttctgat caactctcaa gtccgtccag 1860 agcagttcgc tcatggagtc ecggacctgg cgctgaagga catcgcctgc agcgaggccc 1920 tcctggagcg cttcatcatc ttcagccaga gacgcggggc aaagacggtg cgctatgccc 1980 tgtgctccct ctcccagggc accctacagt ggatagaaga cactctgtat gccaacgtgg 2040 acttcttcaa gctcttccgt gtgcttccca cactcctaga Cagccgttct caaggtatca 2100 atctgagatc ttggggagga atattatctg atatgtcacc aagaattcaa gagtttatcc 2160 atcggccgag tatgqaggac ttgctgtggg tgaccaggcc cctcatgcag aatggtggtc 2220 cagagacctt tacaaagctg atgggcatcc tgtctgacct cctgtgtggc taccccgagg 2280 gaggtggctc tcgggtgctc tccttcaact ggtatgaaga caataactat aaggcctttc 2340 tggggattga ctccacaagg aaggatccta tctattctta tgacagaaga acaacatcct 2400 tttgtaatgc attgatccag agoctggagt caaatccttt aaccaaaatc gottggaggg 2460 cggcaaagcc tttgctgatg ggaaaaatcc tgtacactcc tgattcacct gcagcacgaa 2520 ggatactgaa gaatgccaac tcaacttttg aagaactgga acacgttagg aagttggtca 2580 aagcctggga agaagtaggg acccagatct ggtacttctt tgacaacagc acacagatga 2640 acatgatcag agataccctg gggaacccaa cagtaaaaga ctttttgaat aggcagcttg 2700 gtgaagaagg tattactgct gaagccatcc taaacttcct ctacaagggc cctcgggaaa 2760 gccaggctga cgacatggcc aacttcgact ggagggacat atttaacatc actgatcgca 2820 ccctccgcct tgtcaatcaa tacctggagt gcttggtcct ggataagttt gaaagctaca 2880 atgatgaaac tcagctcacc caacgtgecc tctctctact ggaggaaaac atgttetggg 2940 ccggagtggt attccctgac atgtatccct ggaccagctc tctaccaccc cacgtgaagt 3000 ataagatocg aatggacata gacgtggtgg agaaaaccaa taagattaaa gacaggtatt 3060 gggattctgg tcccagagct gatcccgtgg aagatttccg gtacatctgg ggcgggtttg 3120 cctatctgca ggacatggtt gaacagggga tcacaaggag ccaggtgcag gcggaggctc 3180 cagttggaat ctacctccag cagatgccct acccctgctt cgtggacgat tctttcatga 3240 tcatcctgaa ccgctgtttc cctatcttca tggtgctggc atggatctac tctgtctcca 3300 tgactgtgaa gagcatcgtc ttggagaagg agttgcgact gaaggagacc ttgaaaaatc 3360 agggtgtctc caatgcagtg atttggtgta ectggttcct ggacagcttc tccatcatgt 3420 cgatgagcat cttcctcctg acgatattca tcatgcatgg aagaatccta cattacagcg 3480 acccattcat cctcttcctg ttcttgttgg ctttctccac tgccaccatc atgctgtgct 3540 ttctgctcag caccttcttc tccaaggcca gtctggcagc agcctgtagt ggtgtcatct 3600 atttcaccct ctacctgcca cacatcctgt gettcgcctg gcaggaccgc atgaccgctg 3660 agctgaagaa ggctgtgagc ttactgtctc cggtggcatt tggatttggc actgagtacc 3720 tggttcgctt tgaagagcaa ggcctggggc tgcagtggag caacatcggg aacagtccca 3780 cggaagggga cgaattcagc ttcctgctgt ccatgcagat gatgctcctt gatgctgctg 3840 tctatggctt actcgcttgg taccttgatc aggtgtttcc aggagactat ggaaccccac 3900 ttccttggta ctttcttcta caagagtcgt attggcttgg cggtgaaggg tgttcaacca 3960 gagaagaaag agccctggaa aagaccgagc cqctaaqaga ggaaacggag gatccagagc 4020 acccagaagg aatacacgac tccttctttg aacgtgagca tccagggtgg gttcctgggg 4080 tatgcgtgaa gaatctggta aagatttttg agccctgtgg ccggcoagct gtggaccgtc 4140 tgaacatcac cttctacgag aaccagatca ccgcattcct gggccacaat ggagctggga 4200 aaaccaccac cttgtaagta tcaaggttac aagacaggtt taaggagacc aatagaaact 4260 gggcttgtcg agacagagaa gactcttgcg tttctgggat tttgccgatt tcggcctatt 4320 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 4380 ttataatttc aggtggcatc tttccaattg aggaacccct agtgatggag ttggccactc 4440 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg 4500 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag 4540 <210> 12 <211> 3978

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 12

gtccatcctg acgggtctgt tgccaccaac ctctgggact gtgctcgttg ggggaaggga 6Q cattgaaacc agcctggatg cagtccggca gagccttggc atgtgtccac agcacaacat 120 cctgttccac cacetcacgg tggctgagca catgctgttc tatgcccagc tgaaaggaaa 180 gtcccaggag gaggcccagc tggagatgga agccatgttg gaggacacag gcctccacqa 240 caagcggaat gaagaggctc aggacctatc aggtggcatg cagagaaagc tgtcggttgc 300 cattgccttt gtgggagatg ccaaggtggt gattctggac gaacccacct ctggggtgga 360 cccttactcg agacgctcaa tctgggatct gctcctgaag tatcgctcag gcagaaccat 420 catcatgtcc actcaccaca tggacgaggq cgacctcctt ggggaccgca ttgccatcat 480 tgcccaggga aggctctact gctcaggcac cccactcttc ctgaagaact gctttggcac 540 aggcttgtac ttaaccttgg tgcgcaagat gaaaaacatc cagagccaaa ggaaaggcag 600 tgaggggacc tgcagctgct cgtctaaggg tttctccacc acgtgtccag cccacgtcga 660 tgacctaact ccagaacaag tcctggatgg ggatgtaaat gagctgatgg atgtagttct 720 ccaccatgtt ccagaggcaa agctggtgga gtgcattggt caagaactta tcttccttct 780 tccaaataag aacttcaagc acagagcata tgccagcctt ttcagagagc tggaggagac 840 gctggctgac cttggtctca gcagttttgg aatttctgac actcccctgg aagagatttt 900 tctgaaggtc acggaggatt ctgattcagg acctctgttt gcgggtggcg ctcagcagaa 960 aagagaaaac gtcaaccccc gacacccctg cttgggtccc agagagaagg ctggacagac 1020 accccaggac tccaatgtct gctccccagg ggcgcCggct gctcacCcag agggccagcc 1080 tcccccagag ccagagtgcc caggcccgca gctcaacacg gggacaeagc tggtcctcca 1140 gcatgtgcag gcgctgctgg tcaagagatt ecaacacacc atccgcagcc acaaggactt 1200 cctggcgcag atcgtgctcc cggctaCctt tgtgtttttg gctctgatgc tttctattgt 1260 tatccctcct tttggcgaat accccgcttt gacccttcae ccctggatat atgggcagca 1320 gtacaccttc ttcagcatgg atgaaccagg cagtgagcag ttcacggtac ttgcagacgt 1380 cctcctgaat aagccaggct ttggcaaccg ctgcctgaag gaagggtggc ttccggagta 1440 cccctgtggc aactcaacac cctggaagac tccttctgtg tccccaaaca tcacccagct 1500 gttccagaag cagaaatgga cacaggtcaa cccttcacca tcctgcaggt gcagcaccag 1560 ggagaagctc accatgctgc cagagtgccc cgagggtgcc gggggcctcc cgccccccca 1620 gagaacacag cgcagcacgg aaattctaca agacctgacg gacaggaaca tctccgactt 1680 cttggtaaaa acgtatcctg ctcttataag aagcagctta aagagcaaat tctgggtcaa 1740 tgaacagagg tatggaggaa tttccattgg aggaaagcto ccagtcgtcc ccatcacggg 1800 ggaagcactt gttgggtttt taagcgacct tggccggatc atgaatgtga gcgggggccc 1860 tatcactaga gaggcctcta aagaaatacc tgatttcctt aaacatctag aaactgaaga 1920 caacattaag gtgtggttta ataacaaagg ctggcatgcc ctggtcagct ttctcaatgt 1980 ggcccacaac gccatcttac gggccagcct gcetaaggac agaagccccg aggagtatgg 2040 aatcaccgtc attagccaac ccctgaacct gaccaaggag cagctctcag agattacagt 2100 gctgaccact tcagtggatg ctgtggttgc catctgcgtg attttctcca tgtccttcgt 2160 cccagccagc tttgtccttt atttgatcca ggagcgggtg aacaaatcca agcacctcca 2220 gtttatcagt ggagtgagcc ccaccaccta ctgggtaacc aacttcctct gggacatcat 2280 gaattattcc gtgagtgctg ggctggtggt gggcatcttc atcgggtttc agaagaaagc 2340 ctacacttct ccagaaaacc ttcctgccct tgtggcactg ctcctgctgt atggatgggc 2400 ggtcattccc atgatgtacc cagcatcctt cctgtttgat gtccccagca cagcctatgt 2460 ggctttatct tgtgctaatc tgttcatogg catcaaaagc agtgctatta ccttcatctt 2520 ggaattattt gagaataacc ggacgctgct caggttcaac gccgtgctga ggaagctgct 2580 cattgtcttc ccccacttct gcctgggccg gggcctcatt gaccttgcac tgagccaggc 2640 tgtgacagat gtctatgccc ggtttggtga ggagcactct gcaaatccgt tccactggga 2700 cctgattggg aagaacctgt ttgccatggt ggtggaaggg gtggtgtact tcctcctgao 2760 cctgctggtc cagcgccact tcttCctctc ccaatggatt gccgagccca ctaaggagcc 2820 cattgttgat gaagatgatg atgtggctga agaaagacaa agaattatta ctggtggaaa 2880 taaaactgac atcttaaggc tacatgaact aaccaagatt tatccaggca cctccagccc 2940 agcagtggac aggctgtgtg tcggagttcg ccctggagag tgctttggcc tcctgggagt 3000 gaatggtgcc ggcaaaacaa ccacattcaa gatgctcact ggggacacca cagtgacctc 3060 aggggatgcc accgtagcag gcaagagtat tttaaccaat atttctgaag tccatcaaaa 3120 tatgggctac tgtcctcagt ttgatgcaat cgatgagctg ctcacaggac gagaacatct 3180 ttacctttat gcccggcttc gaggtgtacc agcagaagaa atcgaaaagg ttgcaaactg 3240 gagtattaag agcctgggcc tgactgtcta cgccgactgc ctggctggca cgtacagtgg 3300 gggcaacaag cggaaactct ccacagccat cgcactcatt ggctgcccac cgctggtgct 3360 gqtggatgag cccaccacag ggatggaccc ccaggeacge cgcatgctgt ggaacgtcat 3420 cgtgagcatc atcagagaag ggagggctgt ggtcctcaca tcccacagca tggaagaatg 3480 tgaggcactg tgtacccggc tggccatcat ggtaaagggc gcctttcgat gtatgggcac 3540 cattcagcat etcaagtcca aatttggaga tggctatatc gtcacaatga agatcaaatc 3600 ccegaaggac gacetgcttc ctgacctgaa ccctgtggag cagttcttce aggggaactt 3660 cccaggcagt gtgcagaggg agaggcacta caacatgctc cagttccagg tctcctcctc 3720 ctccctggcg aggatcttcc agctcctcct ctcccacaag gacagcctgc tcatcgagga 3780 gtactcagtc acacagacca cactggacca ggtgtttgta aattttgcta aacagcagac 3840 tgaaagtcat gacctccctc tgcaccctcg agctgctgga gccagtcgac aagoccagga 3900 cgactacaaa gaccatgacg gtgattataa agatcatgac atcgactaca aggatgacga 3960 tgacaagtga gcggccgc 3978 <210> 13 <211 > 261

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 13 ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt 60 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa 120 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg 180 agatgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa atcgataagg 240 atcttcctag agcatggcta c 261 <210> 14 <211>175

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 14 tcactgqtta caaaaccccc ttgcttgaga gtgtggcact ctcecccctg tcgcgttcgc 60 tcgctcgctg gctcgtttgg gggggcgacg gccagagggc cgtcgtctgg cagctctttg 120 agctgccacc cccccaaacg agccagcgag cgagcgaacg cgacaggggg gagag 175 <21O> 15 <211 >4636

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 15 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgccegg ccteagtgag Cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct ggatccggga ttttgccgat ttcggcctat tggttaaaaa atgagetgat 180 ttaacaaaaa tttaaegega attttaacaa aatattaacg tttataattt caggtggcat 240 etttegatag gcacctattg gtcttactga catccacttt geetttetet ccacaggtcc 300 atcctgacgg gtctgttgcc accaacctct gggactgtgc tcgttggggg aagggacatt 360 gaaaccagcc tggatgeagt ccggcagagc cttggcatgt gtccacagca caacatcctg 420 ttecaccacc tcacggtggc tgagcacatg ctgttctatg cccagctgaa aggaaagtcc 480 caggaggagg cccagctgga gatggaagee atgttggagg acacaggcct ccaccacaag 540 cggaatgaag aggctcagga cctatcaggt ggcatgcaga gaaagctgtc ggttgccatt 600 gcctttgtgg gagatgccaa ggtggtgatt ctggacgaac ccacctatgg ggtggacact 660 tactcgagac gctcaatctg ggatctgctc ctgaagtatc gctcaggcag aaccatcatc 720 atgtceactc accacatgga cgaggccgac ctccttgggg accgcattgc catcattgcc 780 cagggaaggc tctactgctc aggcacccca qtcttcqtga agaactgctt tggcacaggc 840 ttgtacttaa ccttggtgcg caagatgaaa aacatcqaga gqcaaaggaa aggcagtgag 900 gggacctgca gctgctcgtc taagggtttc tccaccacgt gtccagccca cgtcgatgac 960 ctaactccag aacaagtcct ggatggggat gtaaatgagc tgatggatgt agttctccac 1020 catgttccag aggcaaagct ggtggagtgc attggtcaag aacttatctt ccttcttcCa 1080 aataagaact tqaagqaqag agqatatgcc agccttttca gagagctgga ggagacgctg 1140 gctgaccttg gtctcagcag ttttggaatt tctgacactc ccctggaaga gatttttctg 1200 aaggtcacgg aggattctga ttcaggacct ctgtttgcgg gtggcgctca gcagaaaaga 1260 gaaaacgtca accqccgaca cccctgcttg ggtcccagag agaaggctgg acagacaccc 1320 caggactcca atgtctgctc cccaggggcg ccggctgctc acccagaggg ccagcctccc 1380 ccagagccag agtgcccagg cccgcagotc aacacgggga cacagctggt cctccagoat 1440 gtgcaggcgc tgctggtcaa gagattocaa cacaccatcc gcagccacaa ggacttcctg 1500 gcgqagatcg tgotcccggc tacctttgtg tttttggctc tgatgctttc tattgttatc 1560 cctccttttg gogaataccc cgctttgacc cttcacccct ggatatatgg gcagqagtac 1620 accttcttca gcatggatga accaggcagt gagcagttca cggtacttgo agacgtcctc 1680 ctgaataagc caggctttgg caaccgctgc qtgaaggaag ggtggcttcc ggagtacccc 1740 tgtggcaact eaacaccctg gaagactcct tctgtgtccc caaacatcac ccagCtgttc 1800 cagaagcaga aatggacaca ggtcaaccct tcaccatcct gcaggtgcag caccagggag 1860 aagctcacca tgctgccaga gtgcoccgag ggtgccgggg gcctcccgcc cccccagaga 1920 acacagcgoa gcacggaaat tctacaagac ctgacggaca ggaacatctc cgacttcttg 1980 gtaaaaacgt atcctgctct tataagaagc agcttaaaga gcaaattctg ggtcaatgaa 2040 cagaggtatg gaggaatttc cattggagga aagctccoag tcgtccccat oacgggggaa 2100 gcacttgttg ggtttttaag cgaccttggc cggatcatga atgtgagcgg gggccctatc 2160 actagagagg cctotaaaga aatacctgat ttccttaaac atctagaaac tgaagacaac 2220 attaaggtgt ggtttaataa caaaggctgg catgccctgg tcagctttct caatgtggcc 2280 caCaacgcca tcttacgggc eagcctgcct aaggacagaa gccccgagga gtatggaatc 2340 accgtcatta gccaacccct gaacctgacc aaggagcagc tctcagagat tacagtgctg 2400 accacttcag tggatgctgt ggttgccatc tgcgtgattt tctccatgtc cttcgtccca 2460 gccagctttg tactttattt gatccaggag cgggtgaaca aatccaagca cctccagttt 2520 atcagtggag tgagccccac caoctaqtgg gtaaecaact tcotctggga catcatgaat 2580 tattccgtga gtgctgggct ggtggtgggc atcttcatcg ggtttcagaa gaaagcctac 2640 acttctccag aaaaccttcc tgcccttgtg gcactgctcc tgctgtatgg atgggcggtc 2700 attcccatga tgtacccagc atccttcctg tttgatgtcc ccagcacagc ctatgtggct 2760 ttatcttgtg ctaatctgtt eatCggcatc aacagcagtg ctattaCctt catcttggaa 2820 ttatttgaga ataaccggac gctgctcagg ttcaacgccg tgctgaggaa gctgctcatt 2880 gtcttccccc acttctgcct gggccggggc ctcattgacc ttgcactgag ccaggctgtg 2940 acagatgtct atgcccggtt tggtgaggag cactctgcaa atccgttcca ctgggacctg 3000 attgggaaga acctgtttgc catggtggtg gaaggggtgg tgtacttcct cctgaccctg 3060 ctggtccagc gccacttctt cctctcccaa tggattgccg agcccactaa ggagcccatt 3120 gttgatgaag atgatgatgt ggctgaagaa agacaaagaa ttattactgg tggaaataaa 3180 actgacatct taaggctaca tgaactaacc aagatttatc caggcacctc cagcccagca 3240 gtggacaggc tgtgtgtcgg agttcgccct ggagagtgct ttggcctcct gggagtgaat 3300 ggtgccggca aaacaaccac attcaagatg ctcactgggg acaccacagt gacctcaggg 3360 gatgccaccg tagcaggeaa gagtatttta accaatattt ctgaagtcca tcaaaatatg 3420 ggctactgtc ctcagtttga tgcaatcgat gagctgctca caggacgaga acatctttac 3480 ctttatgccc ggcttcgagg tgtaccagca gaagaaatcg aaaaggttgc aaactggagt 3540 attaagagcc tgggcctgac tgtctacgcc gactgcctgg etggcacgta cagtgggggc 3600 aacaagcgga aactctccac agccatcgca ctcattggct gcccaccgct ggtgctgctg 3660 gatgagccca ccacagggat ggacccccag gcacgccgca tgctgtggaa cgtcatcgtg 3720 agcatcatca gagaagggag ggctgtggtc ctcacatccc acagcatgga agaatgtgag 3780 gcactgtgta cccggctggc eatcatggta aagggcgcct ttcgatgtat gggcaccatt 3840 cagcatctca agtccaaatt tggagatggc tatatcgtca caatgaagat caaatccccg 3900 aaggacgacc tgcttcctga cctgaaccct gtggagcagt tcttccaggg gaacttccca 3960 ggcagtgtgc agagggagag gcactacaac atgctccagt tccaggtctc ctcctcctcc 4020 ctggcgagga tcttccagct cctcctctcc cacaaggaca gcctgctcat cgaggagtac 4080 tcagtcacac agaccacact ggaccaggtg tttgtaaatt ttgctaaaca gcagactgaa 4140 agtcatgacc tccctctgca ccctcgagct gctggagcca gtcgacaagc ccaggactga 4200 gcggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag 4260 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac 4320 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt 4380 tcagggggag atgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat 4440 cgataaggat cttcctagag catggctacg tagataagta gcatggcggg ttaatcatta 4500 actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 4560 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga 4620 gcgagcgagc gcgcag 4636 <210> 16 <211 > 4431

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 16 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180 aggaagatct tcaatattgg ccattagcca tattattcat tggttatata gcataaatca 240 atattggcta ttggccattg catacgttgt atctatatca taatatgtac atttatattg 300 gctcatgtcc aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat 360 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg 420 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt 480 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac 540 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg 600 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact 660 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt 720 ggcagtacac caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc 780 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc 840 gtaataaccc cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 900 taagcagagc tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc 960 acagttaaat tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca 1020 gaagttggtc gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag 1080 accaatagaa actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta 1140 ttggtcttac tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt 1200 acagctctta aggctagagt acttaatacg actcactata ggctagcctc gagaattcac 1260 gcgtggtacc tctagagtcg acccgggcgg ccgccatggg cttcgtgaga cagatacagc 1320 ttttgctctg gaagaactgg accctgcgga aaaggcaaaa gattcgcttt gtggtggaac 1380 tcgtgtggcc tttatcttta tttctggtct tgatctggtt aaggaatgcc aacccgctct 1440 acagccatca tgaatgccat ttccccaaca aggcgatgcc ctcagcagga atgctgccgt 1500 ggctccaggg gatcttctgc aatgtgaaca atccctgttt tcaaagcccc accccaggag 1560 aatctcctgg aattgtgtca aactataaca actccatctt ggcaagggta tatcgagatt 1620 ttcaagaact cctcatgaat gcaccagaga gccagcacct tggccgtatt tggacagagc 1680 tacacatctt gtcccaattc atggacaccc tccggactca cccggagaga attgcaggaa 1740 gaggaattcg aataagggat atettgaaag atgaagaaac actgacacta tttctcatta 1800 aaaacatcgg cctgtctgac tcagtggtct accttctgat caactctcaa gtccgtccag 1860 agcagttcgc tcatggagtc ecggacctgg cgctgaagga catcgcctgc agcgaggccc 1920 tcctggagcg cttcatcatc ttcagccaga gacgcggggc aaagacggtg cgctatgccc 1980 tgtgctccct ctcccagggc accctacagt ggatagaaga cactctgtat gccaacgtgg 2040 acttcttcaa gctcttccgt gtgcttccca cactcctaga cagccgttct caaggtatca 2100 atctgagatc ttggggagga atattatctg atatgtcacc aagaattcaa gagtttatcc 2160 atcggccgag tatgcaggac ttgctgtggg tgaccaggcc cctcatgcag aatggtggtc 2220 cagagacctt tacaaagctg atgggcatcc tgtctgacct cctgtgtggc taccccgagg 2280 gaggtggctc tcgggtgctc tccttcaact ggtatgaaga caataactat aaggcctttc 2340 tggggattga ctccacaagg aaggatccta tctattctta tgacagaaga acaacatcct 2400 tttgtaatgc attgatccag agcctggagt caaatccttt aaccaaaatc gottggaggg 2460 cggcaaagcc tttgctgatg ggaaaaatcc tgtacactcc tgattcacct gcagcacgaa 2520 ggatactgaa gaatgccaac tcaacttttg aagaactgga acacgttagg aagttggtca 2580 aagcctggga agaagtaggg ccccagatct ggtacttctt tgacaacagc acacagatga 2640 acatgatcag agataccctg gggaacccaa cagtaaaaga ctttttgaat aggcagcttg 2700 gtgaagaagg tattactgct gaagccatcc taaacttcct ctacaagggc cctcgggaaa 2760 gCcaggctga cgacatggcc aacttcgact ggagggacat atttaacatc aCtgatcgca 2820 ccctccgcct tgtcaatcaa tacctggagt gcttggtcct ggataagttt gaaagctaca 2880 atgatgaaac tcagctcacc eaacgtgccc tctctctact ggaggaaaac atgttctggg 2940 ccggagtggt attccctgac atgtatccct ggaccagctc tctaccaccc cacgtgaagt 3000 ataagatccg aatggacata gacgtggtgg agaaaaccaa taagattaaa gacaggtatt 3060 gggattctgg tcccagagct gatcccgtgg aagatttccg gtacatctgg ggcgggtttg 3120 cctatctgca ggacatggtt gaacagggga tcacaaggag ccaggtgcag gcggaggctc 3180 cagttggaat ctacctccag cagatgccct acccctgctt cgtggacgat tctttcatga 3240 tcatcctgaa ccgctgtttc cctatcttca tggtgctggc atggatctac tctgtctcca 3300 tgactgtgaa gagcategtc ttggagaagg agttgcgact gaaggagacc ttgaaaaatc 3360 agggtgtctc caatgcagtg atttggtgta cctggttcct ggacagcttc tccatcatgt 3420 cgatgagcat cttcctcctg acgatattca tcatgcatgg aagaatccta cattacagcg 3480 acccattcat cctcttcctg ttcttgttgg ctttctccac tgccaccatc atgctgtgct 3540 ttctgctcag caccttcttc tccaaggcca gtctggcagc agcctgtagt ggtgtcatct 3600 atttcaccCt ctacctgcca cacatcCtgt gcttcgcctg gcaggaccgc atgaccgctg 3660 agctgaagaa ggctgtgagc ttactgtctc cggtggcatt tggatttggc actgagtacc 3720 tggttcgctt tgaagagcaa ggcctggggc tgcagtggag caacatcggg aacagtccca 3780 cggaagggga cgaattcagc ttcctgctgt ccatgcagat gatgctcctt gatgctgctg 3840 tctatggctt actcgcttgg taccttgatc aggtgtttcc aggagactat ggaaccccac 3900 ttccttggta ctttcttcta caagagtcgt attggcttgg cggtgaaggg tgttcaacca 3960 gagaagaaag agccctggaa aagaccgagc ccctaacaga ggaaacggag gatccagagc 4020 acccagaagg aatacacgac tccttctttg aacgtgagca tccagggtgg gttcctgggg 4080 tatgcgtgaa gaatctggta aagatttttg agccctgtgg ccggccagct gtggaccgtc 4140 tgaacatcac cttctacgag aaccagatca ccgcattcct gggccacaat ggagctggga 4200 aaaccaccac cttgtaagta tcaaggttac aagacaggtt taaggagacc aatagaaact 4260 rrrrcfct Errt· ί~>.·τ ananarrarraa rranEnEEriAi'T 4-/-i 4-/-i a a 4-4- rrccrcfc a ι-ιππη l-arrErtaErirra o.y »>„ a a ^υί.ν^'^ββ^·» aa^'w^v t-w.^ '-"'ri e ** -a«>^.w gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc 4380 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca g 4431 <210> 17 <211 >4521

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 17 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct gataggcaqc tattggtctt actgacatcc actttgcctt tctctqcaca 180 ggtccatcct gacgggtctg ttgccaccaa cctctgggac tgtgctcgtt gggggaaggg 240 acattgaaac cagcctggat gcagtccggc agagccttgg catgtgtcca cagcacaaca 300 tcctgttcca ccacctcacg gtggctgagc acatgctgtt ctatgcccag ctgaaaggaa 360 agtcccagga ggaggcccag ctggagatgg aagccatgtt ggaggacaca ggcctccacc 420 aoaagoggaa tgaagaggct caggacctat caggtggcat gcagagaaag ctgtcggttg 480 ccattgcott tgtgggagat gccaaggtgg tgattctgga cgaacccacc tctggggtgg 540 accCttactc gagacgctca atctgggatc tgctcctgaa gtatcgCtca ggCagaacca 600 tcatcatgtc cactcaccac atggacgagg ccgacctcct tggggaccgc attgccatca 660 ttgcccaggg aaggctctac tgctcaggca ccccactctt cctgaagaac tgctttggca 720 caggcttgta cttaaccttg gtgcgcaaga tgaaaaacat ccagagccaa aggaaaggca 780 gtgaggggac ctgcagctgc tcgtctaagg gtttctceac cacgtgtcca gcccacgtcg 840 atgacctaac tccagaacaa gtcctggatg gggatgtaaa tgagotgatg gatgtagttc 900 tccaccatgt tccagaggca aagctggtgg agtgcattgg tcaagaactt atcttccttc 960 ttccaaataa gaacttcaag cacagagcat atgccagcct tttcagagag ctggaggaga 1020 cgctggctga ccttggtctc agcagttttg gaatttctga cactcccctg gaagagattt 1080 ttctgaaggt cacggaggat tctgattcag gacctctgtt tgcgggtggc gctcagcaga 1140 aaagagaaaa cgtcaacccc cgacacccct gcttgggtcc cagagagaag gctggacaga 1200 caccccagga ctccaatgtc tgctccccag gggcgccggc tgctcaccca gagggccagc 1260 ctcccccaga gccagagtgc ccaggcccgc agctcaacac ggggacacag ctggtcctcc 1320 agcatgtgca ggcgctgctg gtcaagagat tccaacacac catccgcagc cacaaggact 1380 tcctggcgca gatcgtgctc ccggctacct ttgtgttttt ggctctgatg ctttctattg 1440 ttatccctcc ttttggcgaa taccccgctt tgacccttca cccctggata tatgggcagc 1500 agtaoacctt cttCagcatg gatgaaccag gcagtgagca gtteacggta cttgcagacg 1560 tcctcctgaa taagccaggc tttggcaacc gctgcGtgaa ggaagggtgg cttccggagt 1620 accqctgtgg caactcaaca ccctggaaga ctccttctgt gtccccaaac atcacccagc ±b«u tgttccagaa gcagaaatgg aqacaggtca acccttcacc atcctgcagg tgcagcacca 1740 gggagaagct caccatgctg ccagagtgcc ccgagggtgc ogggggoctc ccgccccccc 1800 agagaacaca gcgcagcacg gaaattctac aagacctgac ggacaggaac atctccgact I860 tcttggtaaa aacgtatcct gctcttataa gaagcagett aaagagcaaa ttctgggtca 1920 atgaacagag gtatggagga atttccattg gaggaaagct cccagtcgtc cccatcacgg 1980 gggaagcact tgttgggttt ttaagcgacc ttggccggat catgaatgtg agcgggggcc 2040 ctatcactag agaggcctct aaagaaatac ctgatttcct taaacatcta gaaactgaag 2100 acaacattaa ggtgtggttt aataacaaag gctggcatgc cctggtcagc tttctcaatg 2160 tggcccacaa cgccatetta cgggccagcc tgcctaagga cagaagcccc gaggagtatg 2220 gaatcaccgt cattagccaa cccctgaacc tgaccaagga gcagctatca gagattacag 2280 tgctgaccac ttcagtggat gctgtggttg ccatctgcgt gattttctcc atgtccttcg 2340 tcccagccag ctttgtcctt tatttgatcc aggagcgggt gaacaaatcc aagcacctcc 2400 agtttatcag tggagtgagc cccaqcacct aetgggtaac caacttcctc tgggacatca 2460 tgaattattc cgtgagtgct gggctggtgg tgggcatctt catcgggttt cagaagaaag 2520 cctacacttc tccagaaaac cttcctgccc ttgtggcact gctcctgctg tatggatggg 2580 cggtcattcc catgatgtac ccagcatcct tcctgtttga tgtccccago acagcctatg 2640 tggctttatc ttgtgctaat ctgttcatcg gcatcaacag cagtgctatt accttcatct 2700 tggaattatt tgagaataac cggacgctgc tcaggttcaa cgccgtgctg aggaagctgc 2760 tcattgtctt cccccacttc tgcctgggcc ggggcctcat tgaccttgca ctgagccagg 2820 ctgtgacaga tgtetatgcc cggtttggtg aggagcactc tgcaaatccg ttccactggg 2880 acctgattgg gaagaacctg tttgccatgg tggtggaagg ggtggtgtac ttcctcctga 2940 ccctgctggt ccagcgccac ttcttcctct cccaatggat tgccgagccc actaaggagc 3000 coattgttga tgaagatgat gatgtggctg aagaaagaca aagaattatt actggtggaa 3060 ataaaactga catcttaagg ctacatgaac taaccaagat ttatccaggc acctccagcc 3120 cagcagtgga caggctgtgt gtcggagttc gcoctggaga gtgctttggc ctcctgggag 3180 tgaatggtgc cggcaaaaca accacattca agatgctoac tggggacacc aoagtgacct 3240 caggggatgc caccgtagca ggoaagagta ttttaaccaa tatttctgaa gtccatcaaa 3300 atatgggcta ctgtcctcag tttgatgcaa tcgatgagct gctcacagga cgagaacatc 3360 tttaccttta tgcccggctt cgaggtgtac cagcagaaga aatcgaaaag gttgcaaact 3420 ggagtattaa gagoctgggc ctgactgtct acgccgactg cctggctggc acgtacagtg 3480 ggggcaacaa gcggaaactc tccacagcca tcgcactcat tggctgccca ccgctggtgc 3540 tgctggatga gcccaccaca gggatggacc cccaggcacg ccgcatgctg tggaacgtca 3600 tcgtgagcat catcagagaa gggagggctg tggtcctcae atcccacagc atggaagaat 3660 gtgaggcact gtgtacccgg ctggccatca tggtaaaggg ogcctttcga tgtatgggca 3720 ccattcagca tctcaagtcc aaatttggag atggctatat cgtcacaatg aagatcaaat 3780 ccccgaagga cgacctgctt cctgacctga accctgtgga gcagttcttc caggggaact 3840 tccqaggcag tgtgcagagg gagaggcact acaacatgct ccagttccag gtctcctcct 3900 cctccctggc gaggatcttc cagctcctcc tctcccacaa ggacagcctg ctcatcgagg 3960 agtactcagt cacacagacc acactggacc aggtgtttgt aaattttgct aaacagcaga 4020 ctgaaagtca tgaectccct ctgcaccctc gagctgctgg agccagtcga caagcccagg 4080 actgagcggc cgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca 4140 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt 4200 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt 4260 caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt 4320 aaaatcgata aggatcttcc tagagcatgg ctacgtagat aagtagcatg gcgggttaat 4380 cattaactac aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc 4440 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc 4500 agtgagcgag cgagcgcgca g 4521 <210> 18 <211> 175

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 18 ctctcccccc tgtcgcgttc gctcgctcgc tggctcgttt gggggggtgg cagctcaaag 60 agctgccaga cgacggccCt ctggccgtcg cccccccaaa cgagccagcg agcgagcgaa 120 cgcgacaggg gggagagtgc cacactctca agcaaggggg ttttgtaagc agtga 175 <210> 19 <211 >400

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 19 gctagcgtgc cacctggtcg acattgatta ttgactagtt attaatagta atcaattacg 60 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc 120 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc 180 atagtaacgc caatagggac tttccattga cgtcaatggg tggactattt acggtaaact 240 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat 300 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact 360 tggcagtaca tctacgtatt agtcatcgct attaccatgg 400 <210> 20 <211 >277

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 20 tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa 60 ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg 120 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg 180 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg 240 cggcggccct ataaaaagcg aagcgcgcgg cgggcgg 277 <210> 21 <211> 133

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400>21 gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga 60 cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc 120 tttctctcca cag 133 <210> 22 <211 > 3108

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 22 atggtgattc ttcagcaggg ggaccatgtg tggatggacc tgagattggg gcaggagttc 60 gacgtgccca tcggggcggt ggtgaagctc tgcgactctg ggcaggtcca ggtggtggat 120 gatgaagaca atgaacactg gatctctccg cagaacgeaa cgcacatcaa gcctatgcac 180 cccacgtcgg tccacggcgt ggaggacatg atccgcctgg gggacctcaa cgaggcgggc 240 atcttgcgca acctgcttat ccgctaccgg gaccacctca tctacacgta tacgggctcc 300 atcctggtgg ctgtgaaccc ctaccagctg ctctccatct actcgccaga gcacatccgc 360 cagtatacca acaagaagat tggggagatg cccccccaca tctttgccat tgctgacaac 420 tgctacttca acatgaaacg caaqagccga gaccagtgct gcatcatcag tggggaatct 480 ggggccggga agacggagag cacaaagctg atcctgcagt tcctggcagc catcagtggg 540 cagcactcgt ggattgagca gcaggtcttg gaggccaccc ceattctgga agcatttggg 600 aatgccaaga ccatcCgcaa tgacaactca agccgtttcg gaaagtacat cgacatccac 660 ttcaacaagc ggggcgccat cgagggcgcg aagattgagc agtacctgct ggaaaagtca 720 cgtgtctgtc gccaggccct ggatgaaagg aactaccacg tgttctactg catgctggag 780 ggcatgagtg aggatcagaa gaagaagctg ggcttgggcc aggcctctga ctacaactac 840 ttggccatgg gtaactgcat aacctgtgag ggccgggtgg acagccagga gtacgccaac 900 atccgctccg ccatgaaggt gctcatgttc actgacaccg agaactggga gatctcgaag 960 ctcctggctg ccatcctgca cctgggcaac ctgcagtatg aggcacgcac atttgaaaac 1020 ctggatgcct gtgaggttct cttctcccca tcgctggcca cagctgCatc cctgcttgag 1080 gtgaaccccc cagacctgat gagetgcctg actagccgca ccctcatcac ccgcggggag 1140 acggtgtcca ccocactgag cagggaacag gcactggacg tgcgcgacgc cttcgtaaag 1200 gggatctacg ggcggctgtt cgtgtggatt gtggacaaga tcaacgcagc aatttacaag 1260 cctccctccc aggatgtgaa gaactctcgc aggtccatcg gcctcctgga catctttggg 1320 tttgagaact ttgctgtgaa cagctttgag cagctctgca tcaacttcgc caatgagcac 1380 ctgeagcagt tqtttgtgcg gcacgtgttc aagctggagc aggaggaata tgacctggag 1440 agcattgact ggctgcacat cgagttcact gacaaccagg atgccctgga catgattgcc 1500 aacaagccca tgaacatcat ctccctcatc gatgaggaga gcaagttccc caagggcaca 1560 gacaccacca tgttacacaa gctgaactcc cagcacaagc tcaacgccaa ctaeatcccc 1620 cccaagaaca accatgagac ccagtttggc atcaaccatt ttgcaggcat cgtctactat 1680 gagacccaag gcttcctgga gaagaaccga gacaccctgc atggggacat tatccagctg 1740 gtccactcct ccaggaacaa gttcatcaag cagatcttcc aggccgatgt cgccatgggc 1800 gccgagacca ggaagcgctc gcccacactt agcagccagt tcaagcggtc actggagctg 1860 ctgatgcgca cgctgggtgc ctgccagccc ttotttgtgc gatgcatcaa gcccaatgag 1920 ttcaagaagc ccatgctgtt cgaccggcac ctgtgcgtgc gccagctgcg gtactcagga 1980 atgatggaga ccatcogaat ccgccgagct ggctacocca tccgctacag cttcgtagag 2040 tttgtggagc ggtaccgtgt gctgctgcca ggtgtgaagc cggcctacaa gcagggcgac 2100 ctccgcggga cttgccagcg catggctgag gctgtgctgg gcacccacga tgactggcag 2160 ataggcaaaa ccaagatctt tctgaaggac caccatgaca tgctgctgga agtggagcgg 2220 gacaaagcca tcaccgacag agtcatcctc cttcagaaag tcatccgggg attcaaagac 2280 aggtctaact ttctgaagct gaagaacgct gccacactga tccagaggca ctggcggggt 2340 cacaactgta ggaagaacta cgggctgatg cgtctgggct tcctgcggct gcaggocctg 2400 caccgctccc ggaagctgca ccagcagtac cgcctggccc gccagcgcat catccagttc 2460 caggcocgct gccgcgccta tctggtgcgc aaggccttcc gccaccgcct ctgggctgtg 2520 ctcaccgtgc aggcctatgc ccggggcatg atcgcccgca ggctgcacca acgcctcagg 2580 gctgagtatc tgtggcgcct cgaggctgag aaaatgCggc tggcggagga agagaagctt 2640 cggaaggaga tgagcgccaa gaaggccaag gaggaggccg agcgcaagca tcaggagcgc 2700 ctaocccaac taactccrtaa aaacactaaa caaaaactaa aaaaaaaaaa oaccoctEda 2760 cggaagaagg agctcctgga gcagatggaa agggcccgcc atgagcctgt caatcactca 2820 gacatggtgg acaagatgtt tggcttcctg gggacttcag gtggcctgcc aggccaggag 2880 ggccaggcac ctagtggctt tgaggacctg gagcgagggc ggagggagat ggtggaggag 2940 gacctggatg cagccctgcc cctgcctgac gaggatgagg aggacctctc tgagtataaa 3000 tttgccaagt tcgcggccac ctacttccag gggacaacta cgcactccta cacccggcgg 3060 ccactcaaac agccactgct ctaccatgac gacgagggtg accagctg 3108 <210> 23 <211 >4577

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 23 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180 aggaagatcc taatcgggaa ttcgccctta agctagcgtg ccacctggtc gacattgatt 240 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga 300 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg 360 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg 420 acgtcaatgg gtggactatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca 480 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc 540 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc 600 tattaccatg ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct 660 ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg 720 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg 780 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg 840 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggc tgcagaagtt 900 ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat 960 agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc 1020 ttactgacat ccactttgcc tttctctcca caggtgtcca ggcggccgcc atggtgattc 1080 ttcagcaggg ggaccatgtg tggatggacc tgagattggg gcaggagttc gacgtgccca 1140 tcggggcggt ggtgaagctc tgcgactctg ggcaggtcca ggtggtggat gatgaagaca 1200 atgaacactg gatctctccg cagaaegcaa cgcacatcaa gcctatgcac cccacgtcgg 1260 tccacggcgt ggaggacatg atecgcctgg gggacctcaa cgaggcgggc atcttgcgca 1320 acctgcttat ccgctaccgg gaccacetca tctacacgta tacgggctcc atcctggtgg 1380 ctgtgaaccc ctaccagctg ctctccatct actcgccaga gcacatccgc cagtatacca 1440 acaagaagat tggggagatg cccccccaca tctttgccat tgctgacaac tgctacttca 1500 acatgaaacg caacagccga gaccagtgct gcatcatcag tggggaatct ggggccggga 1560 agacggagag cacaaagctg atcctgcagt tcctggcagc catcagtggg cagcactcgt 1620 ggattgagca gcaggtcttg gaggccaccc ccattctgga agcatttggg aatgccaaga 1680 ccatccgcaa tgacaactca agccgtttcg gaaagtacat cgacatccac ttcaacaagc 1740 ggggcgccat cgagggCgcg aagattgagc agtacctgct ggaaaagtca cgtgtctgtc 1800 gccaggccct ggatgaaagg aactaccacg tgttctactg catgctggag ggcatgagtg 1860 aggatcagaa gaagaagctg ggcttgggcc aggcctctga ctacaactac ttggccatgg 1920 gtaactgcat aacctgtgag ggccgggtgg acagccagga gtacgccaac atccgctccg 1980 ccatgaaggt gctcatgttc actgacaccg agaactggga gatctcgaag ctcctggctg 2040 ccatcctgca cctgggcaac ctgcagtatg aggcacgcac atttgaaaac ctggatgcct 2100 gtgaggttct cttctcccca tcgctggcca cagctgcatc cctgcttgag gtgaaccccc 2160 cagacctgat gagctgcctg actagccgca ccctcatcac ccgcggggag acggtgtcca 2220 ccccaetgag cagggaacag gcactggacg tgcgcgacgc cttcgtaaag gggatctacg 2280 ggcggctgtt cgtgtggatt gtggacaaga tcaacgcagc aatttacaag cctccctccc 2340 aggatgtgaa gaactctcgc aggtccatcg gcctcctgga catctttggg tttgagaact 2400 ttgctgtgaa cagctttgag cagctctgca tcaacttcgc caatgagcac ctgcagcagt 2460 tctttgtgcg gcacgtgttc aagctggagc aggaggaata tgacctggag agcattgact 2520 ggctgcacat cgagttcact gacaaccagg atgccctgga catgattgcc aacaagccca 2580 tgaacatcat ctccctcatc gatgaggaga gcaagttccc caagggcaca gacaccacca 2640 tgttacacaa gctgaactcc cagcacaagc tcaacgccaa ctacatcccc cccaagaaca 2700 accatgagac ccagtttggc atcaaccatt ttgcaggcat cgtctactat gagacccaag 2760 gcttcctgga gaagaaccga gacaccctgc atggggacat tatccagctg gtccactcct 2820 ccaggaacaa gttcatcaag cagatcttcc aggccgatgt cgccatgggc gccgagacca 2880 ggaagcgctc gcccacactt agcagccagt tcaagcggtc actggagctg ctgatgcgca 2940 ogctgggtgc ctgccagccc ttctttgtgc gatgcatcaa gcccaatgag ttcaagaagc 3000 ccatgctgtt cgaccggcac ctgtgcgtgc gccagctgcg gtactcagga atgatggaga 3060 ccatccgaat ccgccgagct ggctacccca tccgctacag cttcgtagag tttgtggagc 3120 ggtaccgtgt gctgctgcca ggtgtgaagc cggcctacaa gcagggcgac ctccgcggga 3180 cttgccagcg catggctgag gctgtgctgg gcacccacga tgactggcag ataggeaaaa 3240 ccaagatctt tctgaaggac caccatgaca tgctgctgga agtggagcgg gacaaagcCa 3300 tcaccgacag agtcatcctc cttcagaaag tcatccgggg attcaaagac aggtctaact 3360 ttctgaagct gaagaacgct gccacactga tccagaggca ctggcggggt cacaactgta 3420 ggaagaacta cgggctgatg cgtctgggct tcetgcggct gcaggccctg caccgctccc 3480 ggaagctgca ccagcagtac cgcctggccc gccagcgcat catceagttc caggcccgct 3540 gccgcgccta tctggtgcgc aaggccttcc gccaccgcct ctgggctgtg ctcaccgtgc 3600 aggcctatgc ccggggcatg atcgcccgca ggctgcacca acgcctcagg gctgagtatc 3660 tgtggcgcct cgaggctgag aaaatgcggc tggcggagga agagaagctt cggaaggaga 3720 tgagcgccaa gaaggccaag gaggaggccg agcgcaagca tcaggagcgc ctggcccaga 3780 tggctcgtga ggacgctgag cgggagctga aggagaagga ggccgctcgg cggaagaagg 3840 agctcctgga gcagatggaa agggcccgcc atgagcctgt caatcactca gacatggtgg 3900 acaagatgtt tggcttcctg gggacttcag gtggcctgcc aggccaggag ggccaggcac 3960 ctagtggctt tgaggacctg gagcgagggc ggagggagat ggtggaggag gacctggatg 4020 cagccctgcc cctgcctgac gaggatgagg aggacctctc tgagtataaa tttgccaagt 4080 tcgcggccac ctacttccag gggacaacta cgcactccta cacccggcgg ccactcaaac 4140 agccactgct ctaccatgac gacgagggtg accagctggt aagtatcaag gttacaagac 4200 aggtttaagg agaccaatag aaactgggcrt tgtcgagaca gagaagaCtc ttgcgtttct 4260 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 4320 gcgaatttta acaaaatatt aacgtttata atttcaggtg gcatctttcc aattgaaggg 4380 cgaattccga tcttcctaga gcatggctac gtagataagt agcatggcgg gttaatcatt 4440 aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc 4500 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgeccgggc ggcctcagtg 4560 agcgagcgag cgcgcag 4577 <210> 24 <211> 3540

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 24 gcagccctgg cggtctggat caccatcctc cgcttcatgg gggacctccc tgagcccaag 60 taccacacag ccatgagtga tggcagtgag aagatccctg tgatgaccaa gatttatgag 120 accctgggca agaagacgta caagagggag ctgcaggccc tgcagggcga gggcgaggcc 180 cagctccccg agggccagaa gaagagcagt gtgaggcaca agctggtgca tttgactctg 240 aaaaagaagt ccaagctcac agaggaggtg accaagaggc tgcatgacgg ggagtccaca 300 gtgcagggca acagcatgct ggaggaccgg cccacctcca acctggagaa gctgcacttc 360 atcatcggca atggcatcct gcggccagca ctccgggacg agatctactg ccagatcagc 420 aagcagctga cccacaaccc ctccaagagc agctatgccc ggggctggat tctcgtgtct 480 ctctgcgtgg gctgtttcgc cccctccgag aagtttgtca agtacctgcg gaacttcatc 540 cacgggggcc cgcccggcta cgccccgtac tgtgaggagc gcctgagaag gacctttgtc 600 aatgggacac ggacacagcc gcccagctgg ctggagctgc aggccaccaa gtccaagaag 660 ccaatcatgt tgcccgtgac attcatggat gggaccacca agaccctgct gacggactcg 720 gcaaccacgg ccaaggagct ctgcaacgcg ctggccgaca agatctctct caaggaccgg 780 ttcgggttct ccctctacat tgccctgttt gacaaggtgt cctccctggg cagcggcagt 840 gaccaqgtca tggacgccat ctcccagtgc gagcagtacg ccaaggagca gggcgcccag 900 gagcgcaacg ccccctggag gctcttcttc cgcaaagagg tcttcacgcc ctggcacagc 960 ccctccgagg acaacgtggc caccaacctc atctaccagc aggtggtgcg aggagtcaag 1020 tttggggagt acaggtgtga gaaggaggac gacctggctg agctggcctc ccagcagtac 1080 tttgtagact atggctctga gatgatcctg gagcgcctcc tgaacctcgt gcccacctac 1140 atccccgaCc gcgagatcac gcccctgaag acgctggaga agtgggccca gctggcCatc 1200 gccgcccaca agaaggggat ttatgcccag aggagaactg atgcccagaa ggtcaaagag 1260 gatgtggtca gttatgcccg cttcaagtgg cccttgctct tctccaggtt ttatgaagcc 1320 tacaaattct caggccccag tctcccCaag aacgacgtca tcgtggccgt caactggacg 1380 ggtgtgtact ttgtggatga gcaggagcag gtacttctgg agctgtcctt cccagagatc 1440 atggccgtgt ccagcagcag ggagtgccgt gtctggctct cactgggctg ctctgatctt 1500 ggctgtgctg cgcctcactc aggctgggca ggactgaccc cggcggggcc ctgttctccg 1560 tgttggtcct gcaggggagc gaaaacgacg gcccccagct tcacgctggc eaccatcaag 1620 ggggacgaat acaccttCac ctccagtaat gctgaggaca ttcgtgacCt ggtggtcacc 1680 ttcctagagg ggctccggaa gagatctaag tatgttgtgg ccctgcagga taaccccaac 1740 cccgcaggcg aggagtcagg cttcctcagc tttgccaagg gagacctcat catcctggac 1800 catgacacgg gcgagcaggt catgaactcg ggctgggcca acggcatcaa tgagaggacc 1860 aagcagcgtg gggaattccc caccgactgt gtgtacgtca tgcccactgt caccatgaca 1920 cctcgtgaga ttgtggccct ggtcaccatg actcccgatc agaggcagga cgttgtccgg 1980 ctcttgqagc tgcgaacggc ggagcccgag gtgcgtgcca agccctacac gctggaggag 2040 ttttcctatg actacttcag gcccccaccc aagcacacgc tgagccgtgt catggtgtcc 2100 aaggcccgag gcaaggaccg gctgtggagc cacacgcggg aaccgctcaa gcaggcgctg 2160 ctcaagaagc tcctgggcag tgaggagctc tcgcaggagg cctgcctggc cttcattgct 2220 gtgctcaagt acatgggcga ctacccgtcc aagaggacac gctccgtcaa tgagctcacc 2280 gaccagatct ttgagggtcc cctgaaagcc gagcccctga aggacgaggc atatgtgcag 2340 atcctgaagc agctgaccga caaccacatc aggtacagcg aggagcgggg ttgggagctg 2400 ctctggctgt gcacgggcct tttcccaccc agcaacatcc tcctgcccca cgtgcagcgc 2460 ttcctgcagt cccgaaagca ctgcccactc gccatcgact gcctgcaacg gctccagaaa 2520 gccctgagaa acgggtcccg gaagtaccct ccgcacctgg tggaggtgga ggccatccag 2580 cacaagacca cccagatttt ccacaaggtc tacttccctg atgacactga cgaggccttc 2640 gaagtggagt ccagcaccaa ggccaaggac ttctgccaga acatcgccac caggctgctc 2700 ctcaagtcct cagagggatt cagcctcttt gtcaaaattg cagacaaggt catcagcgtt 2750 cctgagaatg acttcttctt tgactttgtt cgacacttga cagactggat aaagaaagct 2820 cggcqcatca aggacggaat tgtgccctca ctcacctacc aggtgttctt catgaagaag 2880 ctgtggacca ccacggtgcc agggaaggat cccatggccg attccatctt ccactattac 2940 caggagttgc CCaagtatct CCgaggctac cacaagtgca cgcgggagga ggtgctgcag 3000 ctgggggcgc tgatctaeag ggtcaagttc gaggaggaca agtcctactt ccccagcatc 3060 cceaagctgc tgcgggagct ggtgccccag gaccttatcc ggcaggtctc acctgatgac 3120 tggaagcggt ccatcgtcgc ctacttcaac aagcacgcag ggaagtccaa ggaggaggcc 3180 aagctggcct tcctgaagct catcttcaag tggcccacct ttggctcagc cttcttcgag 3240 gtgaagcaaa ctacggagcc aaacttecct gagatcctcc taattgccat caacaagtat 3300 ggggtcagcc tcatcgatcc caaaacgaag gatatcctca ccactcatcc cttcaccaag 3360 atctccaact ggagcagcgg caacacctac ttccacatca cCattgggaa cttggtgcgc 3420 gggagcaaac tgctctgcga gacgtcactg ggctacaaga tggatgacct cctgacttcc 3480 tacattagcc agatgctoac agcoatgagc aaacagcggg gctccaggag cggcaagtga 3540 <210> 25 <211> 215

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 25 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 60 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 120 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 180 gaggattggg aagacaatag caggcatgct gggga 215 <210> 26 <211 >4389

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 26 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgccegg ccteagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatetaegta gccatgctct 180 aggaagateg gaattegccc tttgatcagg gattttgccg atttcggcct attggttaaa 240 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttataat 300 ttcaggtggc atetttegat aggcacctat tggtcttact gacatccact ttgcctttct 360 ctccacaggc agccctggcg gtctggatca ccatcctecg cttcatgggg gacctccctg 420 agcccaagta ccacacagcc atgagtgatg gcagtgagaa gatccctgtg atgaccaaga 480 tttatgagac cctgggcaag aagacgtaca agagggaget gcaggccctg cagggcgagg 540 gcgaggccca gctecccgag ggccagaaga agagcagtgt gaggcacaag ctggtgcatt 600 tgactctgaa aaagaagtcc aagctcacag aggaggtgac eaagaggctg catgacgggg 660 ay LGuacay l y cayyy uaau aycatyuLyy ayyauuyyuL; Gaud-ucaac cuyyayaayu t £.\j tgcacttcat catcggcaat ggcatcctgc ggccagcact ccgggacgag atctactgcc 780 agatcagcaa gcagctgacc cacaacccct ccaagagcag ctatgcccgg ggctggattc 840 tcgtgtctct ctgcgtgggc tgtttcgccc cctccgagaa gtttgtcaag tacctgcgga 900 acttcatcca cgggggcccg cccggctacg ccccgtactg tgaggagcgc ctgagaagga 960 cctttgtcaa tgggacacgg acacagccgc ccagctggct ggagctgcag gccaccaagt 1020 ccaagaagcc aatcatgttg cccgtgacat tcatggatgg gaccaccaag accctgctga 1080 cggactcggc aaccacggcc aaggagctct gcaacgcgct ggccgacaag atctctctca 1140 aggaccggtt cgggttctcc ctctacattg ccctgtttga caaggtgtcc tccctgggca 1200 gcggcagtga ccacgtcatg gacgccatct cccagtgcga gcagtacgcc aaggagcagg 1260 gcgcccagga gcgcaacgcc ccctggaggc tcttcttccg caaagaggtc ttcacgccct 1320 ggcacagccc ctccgaggac aacgtggcca ccaacctcat ctaccagcag gtggtgcgag 1380 gagtcaagtt tggggagtac aggtgtgaga aggaggacga cctggctgag ctggcctccc 1440 agcagtactt tgtagactat ggctctgaga tgatcctgga gcgcctcctg aacctcgtgc 1500 ccacctacat cccCgaocgc gagatcacgC Ccctgaagac gctggagaag tgggcccagc 1560 tggccatcgc cgcccacaag aaggggattt atgcccagag gagaactgat gcccagaagg 1620 tcaaagagga tgtggtcagt tatgcccgct tcaagtggcc cttgctcttc tccaggtttt 1680 atgaagccta caaattctca ggccccagtc tccccaagaa cgacgtcatc gtggccgtca 1740 actggacggg tgtgtacttt gtggatgagc aggagcaggt acttctggag ctgtccttcc 1800 cagagatcat ggccgtgtcc agcagcaggg agtgccgtgt ctggctctca ctgggctgct 1860 ctgatcttgg ctgtgctgcg cctcactcag gctgggcagg actgaccccg gcggggccct 1920 gttctccgtg ttggtcctgc aggggagcga aaacgacggc ccccagcttc acgctggcca 1980 ccatcaaggg ggacgaatac accttcacct ccagtaatgc tgaggacatt cgtgacctgg 2040 tggtcacctt cctagagggg ctccggaaga gatctaagta tgttgtggcc ctgcaggata 2100 accccaaccc cgcaggcgag gagtcaggct tcctcagctt tgccaaggga gacctcatca 2160 tcctggacca tgacacgggc gagcaggtca tgaactcggg ctgggccaac ggcatcaatg 2220 agaggaccaa gcagcgtggg gacttcccca ccgactgtgt gtacgtcatg cccactgtca 2280 ccatgccacc tcgtgagatt gtggccctgg tcaccatgac tcccgatcag aggcaggacg 2340 ttgtccggct cttgcagctg cgaacggcgg agcccgaggt gcgtgccaag ccctacacgc 2400 tggaggagtt ttcctatgac tacttcaggc ccccacccaa gcacacgctg agccgtgtca 2460 tggtgtccaa ggcccgaggc aaggaccggc tgtggagcca cacgcgggaa ccgctcaagc 2520 aggcgctgct caagaagctc ctgggcagtg aggagctctc gcaggaggcc tgcctggcct 2580 tcattgctgt gctcaagtac atgggcgact acccgtccaa gaggacacgc tccgtcaatg 2640 agctcaccga ccagatcttt gagggtcccc tgaaagccga gcccctgaag gacgaggcat 2700 atgtgeagat cctgaagcag ctgacagaca accacatcag gtacagcgag gagcggggtt 2760 gggagctgct ctggctgtgc acgggccttt tcccacccag caacatcctc ctgccccacg 2820 tgcagcgctt cctgcagtcc cgaaagcact gcccactcgc catcgactgc ctgcaacggc 2880 tccagaaagc cctgagaaac gggtcccgga agtaccctcc gcacctggtg gaggtggagg 2940 ccatccagca caagaccacc cagattttcc acaaggtcta cttccctgat gacactgacg 3000 aggccttcga agtggagtcc agcaccaagg ccaaggactt ctgccagaac atcgccacca 3060 ggctgctcct caagtcctca gagggattca gcctctttgt caaaattgca gacaaggtca 3120 tcagcgttcc tgagaatgac ttcttctttg actttgttcg acacttgaca gactggataa 3180 agaaagctcg gcccatcaag gacggaattg tgccctcact cacctaccag gtgttcttca 3240 tgaagaagct gtggaccacc acggtgccag ggaaggatcc catggccgat tccatcttcc 3300 actattacca ggagttgccc aagtatctcc gaggctacca caagtgcacg cgggaggagg 3360 tgctgcagct gggggcgctg atctacaggg tcaagttcga ggaggacaag tcctacttcc 3420 ccagcatccc caagctgctg cgggagctgg tgccccagga ccttatccgg caggtctcac 3480 ctgatgactg gaagcggtcc atcgtcgcct acttcaacaa gcacgcaggg aagtccaagg 3540 aggaggccaa gctggccttc ctgaagctca tcttcaagtg gcccaccttt ggctcagcct 3600 tcttcgaggt gaagcaaact acggagccaa acttccctga gatcctccta attgccatca 3660 acaagtatgg ggtcagcctc atcgatccca aaacgaagga tatcctcacc actcatccct 3720 tcaCcaagat ctccaactgg agcagcggca aCaCctactt Ccacatcacc attgggaact 3780 tggtgcgcgg gagcaaactg ctctgcgaga cgtcactggg ctacaagatg gatgacctcc 3840 tgacttccta cattagccag atgctcacag ccatgagcaa acagcggggc tccaggagcg 3900 gcaagtgacc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg agatctgcct 3960 cgaqtgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 4020 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 4080 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg 4140 attgggaaga caatagcagg catgctgggg actcgagtta agggcgcaat tcccgattag 4200 gatcttccta gageatggct acgtagataa gtagcatggc gggttaatca ttaactacaa 4260 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc 4320 cgggcgacca aaggtcgcqc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg 4380 agcgcgcag 4389 <210> 27 <211 >4468

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 27 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgccegg ccteagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatetaegta gccatgctct 180 aggaagatcc taatcgggaa ttcgccctta agetagegtg ccacctggtc gacattgatt 240 attgactagt tattaatagt aatcaattac ggggtcatta gtteatagee catatatgga 300 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg 360 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg 420 acgtcaatgg gtggactatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca 480 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc 540 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc 600 tattaccatg ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccet 660 ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg 720 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg 780 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg 840 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggc tgcagaagtt 900 ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat 960 agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc 1020 ttactgacat ccactttgcc tttctctcca caggtgtcca ggcggccgcc atggtgattc 1080 ttcagcaggg ggaccatgtg tggatggacc tgagattggg gcaggagttc gacgtgccca 1140 tcggggcggt ggtgaagctc tgcgactctg ggcaggtcca ggtggtggat gatgaagaca 1200 atgaacactg gatctctccg cagaacgeaa cgcacatcaa gcctatgcac cccacgtcgg 1260 tccacggcgt ggaggacatg atccgcctgg gggacctcaa cgaggcgggc atcttgcgca 1320 acctgcttat ccgctaccgg gaccacctca tctacacgta tacgggctcc atcctggtgg 1380 ctgtgaaccc ctaccagctg ctctccatct actcgccaga gcacatccgc cagtatacca 1440 acaagaagat tggggagatg cccccccaca tctttgccat tgctgacaac tgctacttca 1500 acatgaaacg caacagccga gaccagtgct gcatcatcag tggggaatct ggggccggga 1560 agacggagag cacaaagctg atcctgcagt tcctggcagc catcagtggg cagcactcgt 1620 ggattgagca gcaggtcttg gaggccaccc ccattctgga agcatttggg aatgccaaga 1680 ccatccgcaa tgacaactca agccgtttcg gaaagtacat cgacatccac ttcaacaagc 1740 ggggcgccat cgagggcgcg aagattgagc agtacctgct ggaaaagtca cgtgtctgtc 1800 gccaggccct ggatgaaagg aactaccacg tgttctactg catgctggag ggcatgagtg 1860 aggatcagaa gaagaagctg ggcttgggcc aggcctctga ctacaactac ttggccatgg 1920 gtaactgcat aacctgtgag ggccgggtgg acagccagga gtacgccaac atccgctccg 1980 ccatgaaggt gctcatgttc actgacaccg agaactggga gatctcgaag ctcctggctg 2040 ccatcctgca cctgggcaac ctgcagtatg aggcacgcac atttgaaaac ctggatgcct 2100 gtgaggttct cttctcccca tcgctggcca cagctgcatc cctgcttgag gtgaaccccc 2160 cagacctgat gagctgcctg actagccgca ccctcatcac ccgcggggag acggtgtcca 2220 ccccaetgag cagggaacag gcactggacg tgcgcgacgc cttcgtaaag gggatctacg 2280 ggcggctgtt cgtgtggatt gtggacaaga tcaacgcagc aatttacaag cctccctccc 2340 aggatgtgaa gaactctcgc aggtccatcg gcctcctgga catctttggg tttgagaact 2400 ttgctgtgaa cagCtttgag Cagctctgca tcaacttcgc caatgagcac ctgcagcagt 2460 tctttgtgcg gcacgtgttc aagctggagc aggaggaata tgacctggag agcattgact 2520 ggctgcacat cgagttcact gacaaccagg atgccctgga catgattgcc aacaagccca 2580 tgaacatcat ctccctcatc gatgaggaga gcaagttccc caagggcaca gacaccacca 2640 tgttacacaa gctgaactcc cagcacaagc tcaacgccaa ctacatcccc cccaagaaca 2700 accatgagac ccagtttggc atcaaccatt ttgcaggcat cgtctactat gagacccaag 2760 gcttcctgga gaagaaccga gacaccctgc atggggacat tatccagctg gtccactcct 2820 ccaggaacaa gttcatcaag cagatcttcc aggccgatgt cgccatgggc gccgagacca 2880 ggaagcgctc gcccacactt agcagccagt tcaagcggtc actggagctg Ctgatgcgca 2940 cgctgggtgc ctgccagccc ttctttgtgc gatgcatcaa gcccaatgag ttcaagaagc 3000 ccatgctgtt cgaccggcac ctgtgcgtgc gccagctgcg gtactcagga atgatggaga 3060 ccatccgaat ccgCCgagct ggctacccca tccgctacag cttcgtagag tttgtggagc 3120 ggtaccgtgt gctgctgcca ggtgtgaagc cggcctacaa gcagggcgac ctccgcggga 3180 cttgccagcg catggctgag gctgtgctgg gcacccacga tgactggcag ataggeaaaa 3240 ccaagatctt tctgaaggac caccatgaca tgctgctgga agtggagcgg gacaaagcca 3300 tcaccgacag agtcatcctc cttcagaaag tcatccgggg attcaaagac aggtctaact 3360 ttctgaagct gaagaacgct gccacactga tccagaggca ctggcggggt cacaactgta 3420 ggaagaacta cgggctgatg cgtctgggct tcetgcggct gcaggccctg caccgctccc 3480 ggaagctgca ccagcagtac cgcctggccc gccagcgcat catceagttc caggcccgct 3540 gccgcgccta tctggtgcgc aaggccttcc gccaccgcct ctgggctgtg ctcaccgtgc 3600 aggcctatgc ccggggcatg atcgcccgca ggctgcacca acgcctcagg gctgagtatc 3660 tgtggcgcct egaggetgag aaaatgegge tggeggagga agagaagett eggaaggaga 3720 tgagcgccaa gaaggccaag gaggaggccg agcgcaagca teaggagege ctggcccagc 3780 tggctcgtga ggacgctgag cgggagctga aggagaagga ggccgctcgg eggaagaagg 3840 agctcctgga gcagatggaa agggcccgcc atgagcctgt caatcactca gacatggtgg 3900 acaagatgtt tggettectg gggaettcag gtggcctgcc aggccaggag ggccaggcac 3960 ctagtggctt tgaggacctg gagegaggge ggagggagat ggtggaggag gacctggatg 4020 cagccctgCc cctgcctgac gaggatgagg aggacotctc tgagtataaa tttgecaagt 4080 tcgcggccac ctacttccag gggacaacta cgcactccta cacccggcgg ccactcaaac 4140 agccaetgct ctaccatgac gacgagggtg accagctggt aagtatcaag gttacaagac 4200 aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct 4260 caattgaagg gcgaattccg atcttcctag agCatggcta cgtagataag tagcatggcg 4320 ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg 4380 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg 4440 cggcctcagt gagegagega gcgcgcag 4468 <210> 28 <211 >4268

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 28 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgccegg ccteagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatetaegta gccatgctct 180 aggaagateg gaattegata ggcacctatt ggtcttactg acatecactt tgcctttctc 240 tccacaggca gccctggcgg tctggatcac catcctccgc ttcatggggg acctccctga 300 gcccaagtac cacacagcca tgagtgatgg cagtgagaag atccctgtga tgaccaagat 360 ttatgagacc ctgggcaaga agacgtacaa gagggagctg caggccctgc agggcgaggg 420 cgaggcccag ctccccgagg gccagaagaa gagcagtgtg aggcacaagc tggtgcattt 480 gactctgaaa aagaagtcca agctcacaga ggaggtgacc aagaggctgc atgacgggga 540 gtccacagtg cagggcaaca gcatgctgga ggaccggccc acctccaacc tggagaaget 600 gcacttcatc atcggcaatg gcatcctgcg gccagcactc egggaegaga tctactgcca 660 gatcagcaag cagctgaccc acaacccctc caagagcagc tatgcccggg gctggattct 720 cgtgtctctc tgcgtgggct gtttcgcccc ctccgagaag tttgtcaagt acctgcggaa 780 cttcatccac gggggcccgc ccggctacgc cccgtactgt gaggagegee tgagaaggac 840 ctttgtcaat gggacacgga cacagccgcc cagctggctg gagetgeagg ccaccaagtc 900 caagaagcca atcatgttgc ccgtgacatt catggatggg accaccaaga ccctgctgac 960 ggactcggca accacggcca aggagetetg caacgcgctg gccgacaaga tctctctcaa 1020 ggaCcggttc gggttCtccc tctacattgc cctgtttgac aaggtgtcct ccctgggcag 1080 cggcagtgac cacgtcatgg acgccatctc ccagtgcgag cagtacgcca aggagcaggg 1140 cgcccaggag cgCaacgccc cctggaggct cttCttccgc aaagaggtct tcacgccctg 1200 gcacagcccc tccgaggaca acgtggccac caacctcatc taccagcagg tggtgcgagg 1260 agtcaagttt ggggagtaca ggtgtgagaa ggaggacgac ctggctgagc tggcctccca 1320 gcagtacttt gtagactatg getetgagat gatcctggag cgectcctga acctcgtgcc 1380 cacctaeatc cccgaccgcg agatcacgcc cctgaagacg ctggagaagt gggcccagct 1440 ggccatcgcc gccCacaaga aggggattta tgcccagagg agaaCtgatg cccagaaggt 1500 caaagaggat gtggtcagtt atgcccgctt caagtggccc ttgetettet ccaggtttta 1560 tgaagcctac aaattctcag gceccagtct ccccaagaac gaegteateg tggecgtcaa 1620 ctggacgggt gtgtactttg tggatgagca ggagcaggta Cttctggagc tgtcettccc 1680 agagatqatg gccgtgtcca gcagcaggga gtgccgtgtc tggctctcac tgggctgctc 1740 tgatcttggc tgtgctgcgc ctcactcagg ctgggcagga ctgaccccgg cggggccctg 1800 ttctccgtgt tggtcctgca ggggagcgaa aacgacggcc cccagcttca cgctggccac 1860 catcaagggg gaegaataca ccttcacctc Cagtaatget gaggacattc gtgacctggt 1920 ggtcaccttc ctagaggggc teeggaagag atetaagtat gttgtggccc tgcaggataa 1980 ccccaacccc gcaggcgagg agtcaggctt cctcagcttt gccaagggag acctcatcat 2040 cctggaccat gaqacgggcg agcaggtcat gaactcgggc tgggccaacg gcatcaatga 2100 gaggaccaag cagcgtgggg acttccccac cgactgtgtg tacgtcatgc ccactgtcac 2160 catgccacct cgtgagattg tggccctggt caccatgact cccgatcaga ggcaggacgt 2220 tgtccggctc ttgcagctgc gaacggcgga gcccgaggtg cgtgccaagc cctacacgct 2280 ggaggagttt tcctatgact acttCaggcc cccaccCaag cacacgctga gccgtgtcat 2340 ggtgtccaag gcccgaggca aggaccggct gtggagccac acgcgggaac cgctcaagca 2400 ggcgctgctc aagaagctcc tgggcagtga ggagctctcg caggaggcct gcctggcctt 2460 cattgctgtg ctcaagtaca tgggcgacta cccgtccaag aggacacgct ccgtcaatga 2520 gctcaccgac cagatctttg agggtcccct gaaagccgag cccctgaagg acgaggcata 2580 tgtgcagatc ctgaagcagc tgaccgacaa ccacatcagg tacagcgagg agcggggttg 2640 ggagctgctc tggctgtgca egggcctttt cccacccagc aacatcctcc tgccccacgt 2700 gcagcgcttc ctgcagtccc gaaagcactg cccactcgcc atcgactgcc tgcaacggct 2760 ccagaaagcc ctgagaaacg ggtcccggaa gtaccctccg cacctggtgg aggtggaggc 2820 catccagcac aagaccaccc agattttcca caaggtctac ttccctgatg acactgacga 2880 ggccttcgaa gtggagtcca gcaccaaggc caaggacttc tgccagaaca tcgccaccag 2940 gctgctcctc aagtcctcag agggattcag cctctttgtc aaaattgcag acaaggtcat 3000 cagcgttcct gagaatgact tcttctttga ctttgttcga cacttgacag actggataaa 3060 gaaagctcgg cccatcaagg acggaattgt gccctcactc acctaccagg tgttcttcat 3120 gaagaagctg tggaccacca cggtgccagg gaaggatccc atggccgatt ccatcttcca 3180 ctattaccag gagttgccca agtatctccg aggctaccac aagtgcacgc gggaggaggt 3240 gctgcagctg ggggcgctga tctacagggt caagttcgag gaggacaagt cctacttccc 3300 cagcatcccc aagctgctgc gggagctggt gccccaggac cttatceggc aggtctcacc 3360 tgatgactgg aagcggtcca tcgtcgccta cttcaacaag cacgcaggga agtccaagga 3420 ggaggccaag ctggccttcc tgaagctcat cttcaagtgg cccacctttg gctcagcctt 3480 cttcgaggtg aagcaaacta cggagccaaa cttccctgag atcctcctaa ttgccatcaa 3540 caagtatggg gtcagcctca tcgatcccaa aacgaaggat atcctcacca ctcatccctt 3600 caccaagatc tccaactgga gcagcggcaa cacctacttc cacatcacca ttgggaactt 3660 ggtgcgcggg agcaaactgc tctgcgagac gtcactgggc tacaagatgg atgacctcct 3720 gacttcctac attagccaga tgctcacagc catgagcaaa cagcggggct ccaggagcgg 3780 caagtgaccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcga gatctgcctc 3840 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac 3900 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg 3960 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga 4020 ttgggaagac aatagcaggc atgctgggga ctcgagttaa gggcgcaatt cccgattagg 4080 atcttcctag agcatggcta cgtagataag tagcatggcg ggttaatcat taactacaag 4140 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc 4200 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga 4260 gcgcgcag 4268 <210> 29 <211 >278

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 29 gtgatcctag gtggaggccg aaagtacatg tttcgcatgg gaaccccaga ccctgagtac 60 ccagatgact acagccaagg tgggaccagg ctggacggga agaatctggt gcaggaatgg 120 ctggcgaagc gccagggtgc ccggtacgtg tggaaccgca ctgagctcat gcaggcttcc 180 ctggacccgt ctgtgaccca tctcatgggt ctctttgagc ctggagacat gaaatacgag 240 atccaccgag actccacact ggacccctcc ctgatgga 278 <210> 30 <211 > 66

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 30 gaqtacaaag accatgacgg tgattataaa gatcatgaca tcgactacaa ggatgacgat 60 gacaag 66 <210> 31 <211 > 30

<212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 31 atgtatgatg ttcctgatta tgctagcctc 30

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

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Claims (14)

EFFECTIVE DELIVERY OF BIG GENES THROUGH DUAL-AAV VECTORS A dual construct system for expressing the coding sequence for a gene of interest in a host cell, the coding sequence consisting of a 5 'end portion and a 3' end portion, comprising a dual plasmid comprising a The 5 'to 3' direction comprises: - an AAV 5 'inverted terminal repeat (5' ITR) sequence; - a promoter sequence; - the 5 'end portion of the coding sequence, the 5' end portion operably associated with and under the control of the promoter; - a nucleic acid sequence for a splice donor signal; and - an AAV 3 'inverted terminal repeat (3' ITR) sequence; and b) another plasmid comprising in a 5'-3 'direction: - an AAV 5' inverted terminal repeat (5'-ITR) sequence; a nucleic acid sequence for a splice acceptor signal; - the 3 'end of the coding sequence; a polyadenylation signal nucleic acid sequence; and - an AAV 3 'inverted terminal repeat (3' ITR) sequence, wherein the first plasmid further comprises a nucleic acid sequence for a recombinogenic region at the 5 'position of the AAV 3' ITR of the first plasmid and at the 3 'position of the nucleic acid sequence of the splice donor signal, and wherein the second plasmid further comprises the nucleic acid sequence of the recombinogenic region at the 3' position of the AAV5 'ITR of the second plasmid and at the 5' position of the nucleic acid sequence of the splice donor signal, is a recombinogenic region of an F1 phage consisting of the sequence: GGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAA CGC GAATTTTAACAAAAT (SEQ ID No. 3) or a fragment thereof which retains the SEB property of the SE 3, wherein the coding sequence after insertion of the first plasmid and second plasmid into the host cell reconstitutes by the splice donor and splice acceptor signals. The dual construction system of claim 1, wherein the nucleotide sequence of the ITRs originates from the same AAV serotype or from different AAV serotypes, preferably wherein the 3 'ITR of the first plasmid and the 5' ITR of the second plasmid are from the same AAV serotype, preferably where the 5 'ITR and 3' ITR in the first plasmid and the 5 'ITR and 3' ITR in the second plasmid are respectively from different AAV serotypes, preferably where the 5 'ITR of the first plasmid and the 3' ITR of the second plasmid are from different AAV serotypes. 3. Dual Assembly system according to any one of the preceding claims, wherein the nucleic acid sequence of the splice donor signal consists of the sequence: GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGA GACA GAGAAGACTCTTGCGTTTCT (SEQ ID No. 1), and / or wherein the nucleic acid sequence of splejsningsacceptorsignalet consists of the sequence GATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG (SEQ ID No. 2) . A dual construction system according to any one of the preceding claims, wherein the coding sequence is a nucleotide sequence encoding a protein capable of correcting an inherited retinal degeneration. The dual construction system of claim 4, wherein the coding sequence is selected from the group consisting of: ABCA4, MY07A, CEP290, CDH23, EYS, USH2a, GPR98 or ALMS1. A viral dual adeno-associated virus (AAV) vector system comprising: a) a first viral AAV vector containing the first plasmid as defined in any one of claims 1 to 5, and b) a a second viral AAV vector containing the second plasmid as defined in any one of claims 1 to 5. The viral dual-AAV vector system of claim 6, wherein the adeno-associated virus (AAV) vectors are selected from the same or different AAV serotypes, preferably wherein the adeno-associated virus is selected from serotype 2, serotype 8, serotype 5, serotype 7, or serotype 9. A host cell comprising the viral dual vector system according to any one of claims 6 to 7. A dual construction system according to any one of claims 1 to 5, a viral dual vector system according to any of claims 6 to 7, or a host cell according to claim 8 for medical use, preferably for use in gene therapy. A dual construction system, viral dual vector system, host cell according to claim 9 for use in treating and / or preventing a pathology or disease characterized by a retinal degeneration, preferably where the retinal degeneration is inherited, preferably where the pathology or disease is selected from the group consisting of: retinitis pigmentosa, liver congenital amaurosis (LCA), Stargardt disease, Usher disease, Alstrom syndrome, a disease caused by a mutation in the ABCA4 gene. A pharmaceutical composition comprising the dual construction system of any one of claims 1 to 5, the viral dual vector system of any of claims 6 to 7 or the host cell of claim 8, and a pharmaceutically acceptable vehicle. 12. Nucleic acid consisting of SEQ ID NO. 3 or a fragment thereof which retains the recombinogenic property of SEQ ID NO. 3, for use as a recombinogenic region. A in vitro method for inducing genetic recombination, which method utilizes the sequence consisting of SEQ ID NO. 3 or a fragment thereof which retains the recombinogenic property of SEQ ID NO. Third 14. SEQ ID NO. 3 or a fragment thereof which retains the recombinogenic property of SEQ ID NO. 3, for use in a treatment method, preferably by gene therapy, the method of which induces genetic recombination.